CA3208118A1

CA3208118A1 - Treating diseases and improving nucleic acid delivery

Info

Publication number: CA3208118A1
Application number: CA3208118A
Authority: CA
Inventors: Michael A. Barry; Christopher Y. Chen; Jeffrey D. Rubin; Vincente E. TORRES
Original assignee: Mayo Foundation for Medical Education and Research
Current assignee: Mayo Foundation for Medical Education and Research
Priority date: 2021-01-14
Filing date: 2022-01-14
Publication date: 2022-07-21
Also published as: KR20230146525A; US20240325570A1; WO2022155435A1; JP2024504625A; AU2022208384A1; AU2022208384A9; EP4277994A1

Abstract

This document relates to methods and materials for treating a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a polycystic kidney disease (PKD)). For example, methods and materials that can be used to increase a level of polycystin- 1 (PC-1) polypeptides and/or polycystin-2 (PC-2) polypeptides within a mammal having, or at risk of developing, a polycystic disease) are provided. In some cases, nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal can be administered to a mammal having, or at risk of developing, a polycystic disease to treat the mammal.

Description

TREATING DISEASES AND IMPROVING NUCLEIC ACID DELIVERY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Patent Application Serial No.
63/137,629, filed on January 14, 2021, and U.S. Patent Application Serial No.
63/221,196, filed on July 13, 2021. The disclosures of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.
STATEMENT REGARDING FEDERAL FUNDING
This invention was made with government support under DK090728 and DK123858 awarded by the National Institutes of Health. The government has certain rights in the invention.
SEQUENCE LISTING
This document includes a Sequence Listing that has been submitted electronically as an ASCII text file named 07039-2024W01 5T25.txt. The ASCII text file, created on January 14, 2022, is 269 kilobytes in size. The material in the ASCII text file is hereby incorporated by reference in its entirety.
BACKGROUND
1. Technical Field This document relates to methods and materials for treating a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a polycystic kidney disease (PKD)). For example, methods and materials provided herein can be used to increase a level of polycystin-1 (PC-1) polypeptides and/or polycystin-2 (PC-

2) polypeptides within a mammal having, or at risk of developing, a polycystic disease. In some cases, nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal can be administered to a mammal having, or at risk of developing, a polycystic disease to treat the mammal.
2. Background Information Autosomal dominant polycystic kidney disease (ADPKD) is an inherited progressive disease with a prevalence of approximately one in one thousand live births in which patients develop fluid-filled cysts in their kidneys, losing kidney function, and which can end in kidney failure (see, e.g., Bergmann et al., Nat. Rev. Dis.
Primers., 4(1):50 (2018)).
SUMMARY
ADPKD can be caused by one or more mutations in the PKD1 gene (encoding the PC-1 polypeptide) and/or the PKD2 gene (encoding the PC-2 polypeptide). As such, ADPKD can be treated by gene therapy techniques that can deliver nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal.
However, while many gene therapy vectors can carry the 2.9 kilobase (kb) PKD2 cDNA, most gene therapy vectors and techniques cannot carry the extremely large 12.9 kb PKD1 cDNA.
This document is based, at least in part, on the development of vectors that can be used to deliver nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal. In some cases, this document provides methods and materials for treating a mammal having, or at risk of developing, a polycystic disease (e.g., PKD). For example, nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal can be administered to a mammal having, or at risk of developing, a polycystic disease to treat the mammal. As described herein, adeno-associated virus (AAV) vectors can be used to deliver nucleic acid designed to express a PC-2 polypeptide (e.g., a PKD2 cDNA) to increase the level of PC-2 polypeptides in cells, and helper-dependent adenovirus (HDAd) vectors can be used to deliver nucleic acid designed to express a PC-1 polypeptide (e.g., a PKD1 cDNA) and/or nucleic acid designed to express a PC-2 polypeptide (e.g., a PKD2 cDNA) to increase the level of PC-1 polypeptides and/or PC-2 polypeptides in cells. For example, vectors described herein containing nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be administered to a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a P1(D) to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within the mammal (e.g., to treat the mammal). Also as described herein, one or more AAV vectors can be used to deliver gene therapy components designed for targeted gene activation (e.g., designed for CRISPR-Cas9-based targeted gene activation) of the PKD1 gene and/or the PKD2 gene to upregulate transcription of the PKD1 gene and/or the PKD2 gene to increase the level of PC-1 polypeptides and/or PC-2 polypeptides in cells. For example, one or more nucleic acid molecules designed to express the components of a targeted gene activation system (or the components themselves) designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene can be administered to a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a PKD) to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within the mammal (e.g., to treat the mammal).
This document also provides methods and materials for improving delivery of nucleic acid to a mammal. As described herein, inducing proteinuria in a mammal (e.g., prior to administering a nucleic acid molecule) can improve delivery of nucleic acid (e.g., nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal) to the mammal (e.g., to one or more cells within the mammal).
For example, one or more lipopolysaccharides (LPSs) can be administered to a mammal to induce proteinuria in the mammal to improve delivery of nucleic acid (e.g., nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal) to cells (e.g., kidney cells) within the mammal.
Having the ability to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal provides a unique and unrealized opportunity to treat a polycystic disease such as a PKD.
Having the ability to increase the delivery of nucleic acid to cells within a mammal as described herein can allow for more efficient gene therapy approaches.
In general, one aspect of this document features methods for treating a mammal having a PKD. The methods can include, or consist essentially of, administering to a mammal having a PKD nucleic acid encoding a PC-1 polypeptide or a variant of the PC-1 polypeptide, where the PC-1 polypeptide or the variant is expressed by kidney cells within the mammal. The nucleic acid encoding the PC-1 polypeptide or the variant can be administered to the mammal in the form of a viral vector (e.g., a helper-dependent adenovirus (HDAd) vector). The nucleic acid encoding the PC-1 polypeptide or the variant can be operably linked to a promoter sequence. The promoter sequence can be a human elongation factor la (EF1a) promoter sequence, a chicken 13-actin hybrid (CBh) promoter sequence, a PKD1 promoter sequence, a PKD2 promoter sequence, a

3 cytomegalovirus (CMV) promoter sequence, a Rous sarcoma virus (RSV) promoter sequence, an aquaporin 2 (AQP2) promoter sequence, a gamma-glutamyltransferase (Ggtl) promoter sequence, or a Ksp-cadherin promoter sequence. The method can include identifying the mammal as being in need of a treatment for the PKD.
The mammal can be a human. The PKD can be an autosomal dominant PKD (ADPKD). The method also can include, prior to the administering the nucleic acid, administering a lipopolysaccharides (LPS) to the mammal. The LPS can be administered to the mammal at least 18 hours prior to the administering the nucleic acid. The LPS can be effective to deliver large nucleic acid to the kidney cells in the mammal.
In another aspect, this document features methods for treating a mammal having a PKD. The methods can include, or consist essentially of, administering to a mammal having a PKD nucleic acid encoding a PC-2 polypeptide or a variant of the PC-2 polypeptide, where the PC-2 polypeptide or the variant is expressed by kidney cells within the mammal. The nucleic acid encoding the PC-2 polypeptide or the variant can be administered to the mammal in the form of a viral vector (e.g., an adenovirus-associated virus (AAV) vector). The nucleic acid encoding the PC-2 polypeptide or the variant can be operably linked to a promoter sequence. The promoter sequence can be a EFla promoter sequence, a CBh promoter sequence, a PKD1 promoter sequence, a promoter sequence, a CMV promoter sequence, a RSV promoter sequence, an AQP2 promoter sequence, a Ggtl promoter sequence, or a Ksp-cadherin promoter sequence.
The method can include identifying the mammal as being in need of a treatment for the PKD. The mammal can be a human. The PKD can be an autosomal dominant PKD
(ADPKD). The method also can include, prior to the administering the nucleic acid, administering a lipopolysaccharides (LPS) to the mammal. The LPS can be administered to the mammal at least 18 hours prior to the administering the nucleic acid.
The LPS can be effective to deliver large nucleic acid to the kidney cells in the mammal.
In another aspect, this document features methods for treating a mammal having a PKD. The methods can include, or consist essentially of, administering to a mammal having a PKD: (a) nucleic acid encoding a PC-1 polypeptide or a variant of the polypeptide, where the PC-1 polypeptide or the variant is expressed by kidney cells within the mammal; and (b) nucleic acid encoding a PC-2 polypeptide or a variant of the PC-2 polypeptide, where the PC-2 polypeptide or the variant is expressed by kidney cells

4

5 within the mammal. The nucleic acid encoding the PC-1 polypeptide or the variant can be administered to the mammal in the form of a viral vector (e.g., a HDAd vector). The nucleic acid encoding the PC-1 polypeptide or the variant can be operably linked to a promoter sequence. The promoter sequence can be a EFla promoter sequence, a CBh promoter sequence, a PKD1 promoter sequence, a PKD2 promoter sequence, a CMV
promoter sequence, a RSV promoter sequence, an AQP2 promoter sequence, a Ggtl promoter sequence, or a Ksp-cadherin promoter sequence. The nucleic acid encoding the PC-2 polypeptide or the variant can be administered to said mammal in the form of a viral vector (e.g., an AAV vector). The nucleic acid encoding the PC-2 polypeptide or the variant can be operably linked to a promoter sequence. The promoter sequence can be a a EFla promoter sequence, a CBh promoter sequence, a PKD1 promoter sequence, a promoter sequence, a CMV promoter sequence, a RSV promoter sequence, an AQP2 promoter sequence, a Ggtl promoter sequence, or a Ksp-cadherin promoter sequence.
The nucleic acid encoding the PC-1 polypeptide or the variant and the nucleic acid encoding the PC-2 polypeptide or the variant are administered to the mammal in the form of a viral vector (e.g., a HDAd vector). The nucleic acid encoding the PC-1 polypeptide or the variant can be operably linked to a first promoter sequence, and the nucleic acid encoding the PC-2 polypeptide or the variant can be operably linked to a second promoter sequence. The first promoter sequence and the second promoter sequence can each be independently selected from the group consisting of a EFla promoter sequence, a CBh promoter sequence, a PKD1 promoter sequence, a PKD2 promoter sequence, a CMV
promoter sequence, a RSV promoter sequence, an AQP2 promoter sequence, a Ggtl promoter sequence, and a Ksp-cadherin promoter sequence. The method can include identifying the mammal as being in need of a treatment for the PKD. The mammal can .. be a human. The PKD can be an autosomal dominant PKD (ADPKD). The method also can include, prior to the administering the nucleic acid, administering a lipopolysaccharides (LPS) to the mammal. The LPS can be administered to the mammal at least 18 hours prior to the administering the nucleic acid. The LPS can be effective to deliver large nucleic acid to the kidney cells in the mammal. The method can include identifying the mammal as being in need of a treatment for the PKD. The mammal can be a human. The PKD can be an autosomal dominant PKD (ADPKD). The method also can include, prior to the administering the nucleic acid, administering a lipopolysaccharides (LPS) to the mammal. The LPS can be administered to the mammal at least 18 hours prior to the administering the nucleic acid. The LPS can be effective to deliver large nucleic acid to the kidney cells in the mammal.
In another aspect, this document features methods for treating a mammal having a PKD. The methods can include, or consist essentially of, administering to a mammal having a PKD: (a) nucleic acid encoding a fusion polypeptide including a deactivated Cos (dCas) polypeptide and a transcriptional activator polypeptide; (b) nucleic acid encoding a helper activator polypeptide; and (c) nucleic acid encoding a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene, and (ii) a nucleic acid sequence that can bind the helper activator polypeptide. The dCas polypeptide can be a deactivated Cas9 (dCas9) polypeptide or a deactivated Cas phi (dCas(D) polypeptide. The transcriptional activator polypeptide can be a VP64 polypeptide. The fusion polypeptide can be a dCas9-VP64 fusion polypeptide.
The helper activator polypeptide can be a MS2 polypeptide, a p65 polypeptide, a HSF1 polypeptide, or a VP64 polypeptide. The helper activator polypeptide can include a MS2 polypeptide, a p65 polypeptide, and a HSF1 polypeptide. The nucleic acid (a), the nucleic acid (b), and the nucleic acid (c) can be administered to the mammal in the form of a viral vector. The viral vector can be a HDAd, a lentiviral vector, or an AAV vector.
The nucleic acid (a) can be administered to the mammal in the form of a first viral vector, and the nucleic acid (b) and the nucleic acid (c) can be administered to the mammal in the form of a second viral vector. The first viral vector can be an AAV vector and the second viral vector can be an AAV vector. The nucleic acid (a) can be operably linked to a first promoter sequence, the nucleic acid (b) can be operably linked to a second promoter sequence, and the nucleic acid (c) can be operably linked to a third promoter sequence.
The first promoter sequence, the second promoter sequence, and the third promoter sequence can each independently be selected from the group consisting of a EF1a promoter sequence, a CBh promoter sequence, a CMV promoter sequence, a RSV
promoter sequence, a U6 promoter sequence, an AQP2 promoter sequence, a Ggtl promoter sequence, and a Ksp-cadherin promoter sequence. The method also can include identifying the mammal as being in need of a treatment for the PKD. The mammal can be a human. The PKD can be an ADPKD. The also can include, prior to the administering the nucleic acid, administering a LPS to the mammal. The LPS can be

6 administered to the mammal at least 18 hours prior to the administering the nucleic acid.
The administering the LPS can be effective to deliver large nucleic acid to the kidney cells in the mammal.
In another aspect, this document features methods for delivering nucleic acid to a cell within a mammal. The methods can include, or consist essentially of, (a) administering a proteinuria-inducing agent to a mammal; and (b) administering nucleic acid to the mammal. The mammal can be a human. The proteinuria-inducing agent can be LPS, puromycin, adriamycin, protamine sulfate, cationic albumin, or polycations. The nucleic acid can be from about 0.15 kb to about 36 kb in size. The nucleic acid can have a mass of from about 10 kilodaltons (kDa) to about 50 kDa. The nucleic acid can have a diameter of from about 10 nm to about 26 nm. The method can include administering from about 7 milligrams per kilogram body weight (mg/kg) to about 9 mg/kg of the proteinuria-inducing agent to the mammal. The cell can be a kidney cell, a spleen cell, a lungs cell, or a brain cell. The proteinuria-inducing agent can be administered to the mammal at least 18 hours prior to the administering the nucleic acid. The administering the proteinuria-inducing agent can include intravenous injection. The administering the nucleic acid can include intravenous injection. The administering the proteinuria-inducing agent can include intravenous injection, and the administering the nucleic acid can include intravenous injection.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

7 DESCRIPTION OF THE DRAWINGS
Figures 1A-1D. Diagrams of exemplary in vivo vectors for delivery ofPKD1 and PKD2 cDNAs. Figure lA shows a single HDAd vector including a PKD1 cDNA with additional space for cargo, denoted as "stuffer". Figure 1B shows an AAV
vector including a PKD2 cDNA. Figure 1C shows an HDAd vector including both a PKD1 cDNA and a PKD2 cDNA. ITR = inverted terminal repeat, EFla = human elongation factor la promoter, CBh = chicken 13-actin hybrid promoter. Figure 1D shows alternative HDAd vectors including a PKD1 cDNA and/or a PKD2 cDNA.
Figure 2. A schematic of an exemplary process used to generate triple transduced, stable cell lines expressing Cas9-SAM. LV = lentivirus, Bsd = blasticidin, Hyg =
hygromycin, Zeo = zeocin.
Figure 3. A graph showing fold PKD1 gene expression of human 293 cells transduced to express Cas9-SAM. qRT-PCR was performed with one biological replicate and three technical replicates (n=1). RQ = relative quantitation.
Figure 4. A graph showing fold PKD1 gene expression of human RCTE cells transduced to express Cas9-SAM. qRT-PCR was performed with one biological replicate and three technical replicates (n=1). RQ = relative quantitation.
Figure 5. A graph showing fold Pkdl gene expression of mouse IMCD3 cells transduced to express Cas9-SAM. qRT-PCR was performed with one biological replicate and three technical replicates (n=1). RQ = relative quantitation.
Figures 6A-6D. Diagrams of exemplary vectors for in vivo delivery of Cas9-SAM.

Figure 6A shows a single HDAd vector delivering the entire Cas9-SAM system with additional space for cargo, denoted as "stuffer". Figure 6B shows a single lentiviral vector delivering the entire Cas9-SAM system. Figure 6C shows a dual AAV
vector system for delivering the Cas9-SAM system in two pieces. Figure 6D shows a single AAV vector system for delivering the SAM system based on a newly discovered and smaller Casill protein. ITR = inverted terminal repeat, LTR = long terminal repeat, U6 =
U6 promoter, CMV = human cytomegalovirus promoter, EFla = human elongation factor la promoter, CBh = chicken 13-actin hybrid promoter, P2A = 2A self-cleaving peptide.
Figure 7. A western blot of dCas9VP64 protein from transfected viral vector expression cassettes. All three transfected AAV cassettes and the transfected Ad cassette produced dCas9VP64 protein, which is calculated to have a mass of 168.26 kilodaltons.

8 EFla = human elongation factor 1a promoter, CMV = human cytomegalovirus promoter, FpA = Ad5 Fiber polyadenylation signal, HGHpA = Human growth hormone polyadenylation signal.
Figure 8. Ex vivo luminescent imaging of livers and kidneys after intravenous injection with AAV8, with or without induced proteinuria. Mice were administered either PBS or LPS by intraperitoneal (i.p.) injection or intravenous (i.v.) injection with 1.94e12 genome copies of self-complementary (sc) AAV8-Cre a day later (n=1). Six days after AAV injection, the mice were sacrificed and their livers and kidneys were imaged for luminescence ex vivo. While the liver signals remained consistent, the mouse injected with LPS exhibited greater luminescence from its kidneys than the PBS-injected mouse.
LK = left kidney, RK = right kidney.
Figure 9. Fluorescent imaging of liver and kidney sections after intravenous injection with AAV8, with or without induced proteinuria. The same liver and kidney tissues from Figure 8 were sectioned to view transduced (EGFP+) cells. The livers from both mice appear to be almost entirely transduced after a high dose of the liver tropic AAV8. The kidneys of the LPS-injected mouse shows transduced glomeruli and proximal tubules whereas the kidneys of the PBS-injected mouse show only transduced glomeruli. Arrows point to transduced proximal tubules adjacent to glomeruli.
Figures 10A-10D. Ex vivo liver and kidney luminescence and flow cytometry with a lower dose of AAV8, with or without proteinuria. Figure 10A contains a graph showing no significant difference in liver luminescent between PBS and LPS-injected mice (n=3; p = 0.2000). Figure 10B contains a graph showing that kidneys of LPS-injected mice exhibited significantly more luminescence than those of PBS-injected mice (n=; *p = 0.0260). Figures 10C and 10D contain graphs showing the percent of GFP+
cells in kidneys from Figure 10B that were homogenized, stained, and analyzed by flow cytometry. Figure 10C shows that EpCAM+CD31- (epithelial) cells had a significant increase in transduction (n=6; **p = 0.0022). Figure 10D shows that EpCAM-CD31+
(endothelial) cells showed no significant change in transduction between LPS
and PBS-injected mice (n=6; p = 0.6991).
Figures 11A-11C. Investigation of mice injected i.v. with Ad5-Cre, with or without induced proteinuria. Figure 11A contains exemplary images of bisected kidneys of one PBS/Ad5-Cre mouse and one LPS/Ad5-Cre mouse. Mice were sacrificed and their

9 kidneys were imaged ex vivo (n=3). LPS-injected mouse kidneys exhibiting increased luminescence. Figure 11B contains a graph showing quantitation of ex vivo kidney luminescence. Luminescence significantly increased in LPS-injected mice from PBS-injected mice (n=6 kidneys; **p = 0.0022). Figure 11C contains exemplary fluorescent images of liver and kidney sections. Liver transduction decreased and kidney transduction increased, specifically in the glomeruli, in the LPS-injected mice. Arrows point to increased transduction in glomeruli.
Figures 12A-12B. PC-1 sequences. Figure 12A is a representative nucleic acid sequence that can encode a human PC-1 polypeptide (SEQ ID NO:1). Figure 12B is an to amino acid sequence of a representative human PC-1 polypeptide (SEQ ID
NO: 2).
Figures 13A-13B. PC-2 sequences. Figure 13A is a representative nucleic acid sequence that can encode a human PC-2 polypeptide (SEQ ID NO:3). Figure 13B is an amino acid sequence of a representative human PC-2 polypeptide (SEQ ID NO:4).
Figures 14A-14B. Intravenous delivery of AAV8 in a state of induced proteinuria enhances kidney transduction. Figure 14A. Diagram of experimental scheme. Two month old male luciferase-mT/mG triple reporter mice were administered LPS
intraperitoneally on Day -1 and scAAV intravenously on Day 0. In vivo bioluminescence was assessed daily until peak expression was observed at Day 6. Figure 14B. In vivo bioluminescence at Day 6 followed by ex vivo luminescence of livers and kidneys. n = 1 mouse per group.
Figure 15. Intravenous delivery of multiple AAV serotypes enhances tubule epithelial cell transduction, but not necessarily proximal tubule cell transduction. The same kidneys from Figure 14 were sectioned to examine endogenous mT and mG
fluorescence. Arrows point to examples of transduced non-glomerular (tubular) cells.
While some tubular cell transduction was observed in PBS-injected control mice (left panels), there were increased numbers of these cells in LPS-injected induced proteinuria mice (center panels). No instances of these transduced cells were observed to be counterstained by LTL, a marker of proximal tubule cells (right panels). n = 1 mouse per group.
Figures 16A-16C. Intravenous delivery of scAAV8 in a state of induced proteinuria significantly increases transduction of renal epithelial cells.
Figure 16A.
Three month old male mice were administered an i.p. injection of either PBS or LPS at Day -1 and an i.v. injection of 2.03e11 GC of scAAV8-Cre at Day 0. At Day 6, in vivo luminescence and ex vivo liver luminescence were not significantly different between PBS and LPS-injected groups, although brain luminescence was significantly increased in the LPS-injected group (p = 0.0475 by Welch's t test.). n = 3 mice per group, except for control group where n = 1; error bars are represented by mean with SD. Figure 16B.
Kidneys were bisected with a razor blade to reduce obstruction of luminescence and imaged ex vivo, with the LPS-injected group exhibiting increased luminescence compared to the PBS-injected group. Figure 16C. Ex vivo luminescence from Panel B was quantified and kidneys were subsequently processed for flow cytometry.
Overall, kidneys from LPS-injected mice showed significantly higher ex vivo luminescence and percentage of transduced epithelial cells, but not of transduced endothelial cells (p values obtained using Mann-Whitney test). n = 6 kidneys per group, except for control group where n = 1; error bars are represented by mean with SD.
Figures 17A-17B. AAVrhl 0 does not necessarily increase transduction of tubule epithelial cells during induced proteinuria. Figure 17A. Eight month old female mice were administered an i.p. injection of either PBS or LPS at Day -1 and an i.v.
injection of 1.76ell GC of scAAVrh10-Cre at Day 0. At Day 5, kidneys were processed for flow cytometry. Although there was no difference in transduced CD45- (non-hematopoietic) kidney cells, kidneys of the LPS-injected group had a significant increase in CD45+
(hematopoietic) cells compared to the PBS-injected group (n = 6 kidneys per group).
Figure 17B. Kidney CD45- (non-hematopoietic) cells were separately gated into EpCAM+ CD31- (all epithelial cells), EpCAM- CD31+ (endothelial cells), and EpCAM+
LTL+ and EpCAM+ AQP1+ (two different markers of proximal tubule cells). None of the aforementioned gating strategies showed a significant difference in transduced cells between PBS-injected and LPS-injected groups. n = 6 kidneys per group, except for control group where n = 1; error bars are represented by mean with SD for all panels. p values determined using Mann-Whitney tests for all panels.
Figures 18A-18B. Examination of kidney transduction using a vector with low liver tropism. Figure 18A. Four and a half month old female mice were administered an i.p. injection of PBS or LPS on Day -1 and an i.v. injection of 9.5e10 GC of scAAV1-Cre on Day 0. In vivo bioluminescence was assessed daily until peak expression was observed at Day 6. No significant difference was observed between groups, including a measurement of ex vivo liver luminescence (p values determined using Welch's t test). n = 3 mice per group, except for control group where n = 1. Error bars are represented by mean with error (top left) or mean with SD (top right). Ex vivo kidney luminescence showed that LPS-injected mice had an increased but insignificant amount of luminescence compared to PBS-injected mice as well as LPS and scAAV8-Cre injected mice (p values determined using Mann-Whitney test). n = 6 kidneys per group, lower panels; error bars are represented by mean with SD; scAAV8-Cre data represents the same data shown in Figure 16. Figure 18B. The kidneys analyzed in Panel A were sectioned to observe endogenous mT and mG fluorescence. While mice treated with PBS
and scAAV1-Cre showed transduction primarily in glomeruli (left), mice treated with LPS and scAAV1-Cre showed increased transduction in non-glomerular (tubular) cells (right). Arrows point to examples of transduced glomerular cells (left) or examples of transduced tubule cells (right).
Figures 19A-19C. Induced proteinuria increases adenovirus transduction of the kidney, but strictly in glomeruli. Figure 19A. Four month old mice were administered an i.p. injection of PBS (male mice) or LPS (female mice) on Day -1 and an i.v.
injection of 1 ell vp of Ad5-Cre on Day 0. In vivo bioluminescence was assessed daily until peak expression was observed at Day 5. Luminescence was significantly lower in LPS-injected mice compared to PBS-injected mice (p value determined using Welch's t test; n = 3 mice per group; error bars are represented by mean with error, left), however, ex vivo kidney luminescence was significantly higher in LPS-injected mice compared to PBS-injected mice (p value determined using Mann-Whitney test; n = 6 kidneys per group, except for control group where n = 1; error bars are represented by mean with SD, right).
Figure 19B. Bioluminescent images of the kidneys quantified ex vivo in Panel A. While essentially no luminescence is visible in kidneys from mice injected with PBS, kidneys from mice injected with LPS showed luminescence localized to the renal pelvis region of the kidney. Figure 19C. Kidneys shown in Panel B were sectioned to examine mT
and mG endogenous fluorescence. Yellow arrows point to examples to transduced glomerular cells, which are present sparsely in mice injected with PBS and more frequently in mice injected with LPS. No instances of transduced tubular cells were observed in either group of mice.

Figures 20A-20B. Induced proteinuria increases AAV gene delivery to renal epithelial cells in mice with polycystic kidney disease. Figure 20A. Male Pkd1RC/RC-mT/mG hybrid mice were generated, which have two hypomorphic Pkd1RC alleles and develop autosomal dominant polycystic kidney disease. Nine month old male mice were treated with PBS or LPS via i.p. injection at Day -1 and 1.64e11 GC of scAAV8-Cre via i.v. injection at Day 0. Figure 20B. Mice were sacrificed at Day 6 and their kidneys were sectioned to examine mT and mG endogenous fluorescence. Arrows point to transduced cells. While transduced glomerular cells were observed in PBS-injected mice, transduced tubular cells were observed only in LPS-injected mice (n = 1 mouse for each group).
Figure 21. Diagram modeling vector pharmacokinetics in a state of induced proteinuria. LPS administration results in degradation of podocyte foot processes, effectively increasing the permselectivity of slit diaphragms to an unknown diameter above the natural 10 nm. This change in physiology allows the smaller AAV (25 nm i.d.) to penetrate into adjacent tubule cells while the larger Ad (90 nm i.d.) has increased penetration into glomerular cells but not tubular cells. It is also possible that AAV moved from the vasculature of the kidney to transduce cells of the macula densa.
Figure 22. Example of proteinuria dipsticks used to assess induced proteinuria in mice. Mice administered LPS at Day -1 had a higher indicated level of proteinuria at Day 0 while mice administered PBS had a consistent level of proteinuria from Day -1 to Day 0. It was common for mice administered LPS to have a proteinuria level of greater than 2000 mg/dL the following day.
Figures 23A-23B. Administration of LPS to mice did not affect liver transduction by AAV but did result in renal medullar transduction across several serotypes of AAV.
Figure 23A. Quantification of in vivo luminescence images shown in Figure 14.
Mice that were administered i.p. injections of either PBS or LPS on Day -1 and i.v.
injections of scAAV8-Cre, scAAV9-Cre, or scAAVrh10-Cre on Day 0 had levels of in vivo luminescence that varied by approximately two orders of magnitude on Day 1, but these signals reached approximately the same level by Day 6. Figure 23B. Images of the medulla of kidneys of scAAV8 and scAAV9 injected mice from Figure 15. Both images show that there is transduction in medullar cells in addition to the cortical tubular cells shown earlier.

Figures 24A-24B. Evidence of toxicity associated with combined LPS and AAV
administration. Figure 24A. Liver sections of mice injected with PBS or LPS
followed by high-dose scAAV8-Cre. These sections are from the same mice injected with scAAV8-Cre in Figures 14 and 15. While both livers are entirely transduced by scAAV8-Cre, the liver of the LPS-injected mouse exhibited a globular cell phenotype indicative of toxicity.
Figure 24B. From the mice in Figure 3, mice treated with LPS had significantly increased transduced levels of macrophages in the blood compared to mice treated PBS, indicating an overall increase in macrophage present in the blood after LPS treatment. (p = 0.0475 by Welch's t test.) Figures 25A-25D. Representative flow cytometry plots for mice administered scAAV8-Cre. Plots are from Left Kidney of Mouse #01 Left Kidney (treated with PBS
followed by PBS, in a group of n = 2 kidneys) and Mouse #07 (treated with LPS
followed by scAAV8-Cre, in a group of n = 6 kidneys).
Figures 26A-26C. Representative flow cytometry plots for mice administered scAAVrh10-Cre. Plots are from Left Kidney of Mouse #01 Left Kidney (treated with PBS followed by PBS, in a group of n = 2 kidneys) and Mouse #02 (treated with LPS
followed by scAAVrh10-Cre, in a group of n = 6 kidneys).
Figures 27A-27B. Increased kidney transduction after administration of LPS and Ad5-Cre is negatively correlated with liver transduction. Figure 27A. Example of liver sections of mice injected either with PBS followed by Ad5-Cre or LPS followed by Ad5-Cre. While the liver of the former is fully transduced, the liver of the latter is only partially transduced. Figure 27.) The mice shown are the same mice from Figure injected with LPS followed by Ad5-Cre. In vivo imaging (top) is juxtaposed to corresponding liver section (middle) and ex vivo kidney imaging (bottom). Mice with weaker liver transduction exhibited stronger kidney transduction.
Figure 28. Comparison of liver transduction across various vectors and doses.
While scAAV8, scAAV9, and scAAVrhl 0 fully transduced the liver, scAAV1 only partially transduced the liver. Ad5-Cre fully transduced the liver, which was attenuated when LPS was administered prior to Ad5-Cre.
Figure 29. Livers of mice with polycystic kidney disease were fully transduced by scAAV8-Cre. Livers of mice shown in Figure 20. The livers of these mice were fully transduced when injected with scAAV8-Cre.

Figure 30. A schematic representation of a cre recombinase activated reporter mouse model.
Figures 31A ¨ 31B. Representative images of mice showing luciferase expression. Figure 31A. In vivo luminescent imaging. Figure 31B. Fluorescent imaging of tissue sections.
Figures 32A ¨ 32E. Figure 32A. Fluorescent imaging of kidney sections following transduction with different AAV serotypes. Figure 32B.
Immunostaining of smooth muscle and proximal tubules in kidneys of mice treated with AAV1.
Figure 32C.
Immunostaining of smooth muscle and proximal tubules in kidneys of mice treated with to .. AAV8. Figure 32D. Immunostaining of podocytes, smooth muscle, and proximal tubules in kidneys of mice treated with AAV9. Figure 32E. Immunostaining of endothelium, smooth muscle, and proximal tubules in kidneys of mice treated with AAVrh10.1.
Figure 33. Immunostaining of endothelium in kidneys of mice treated with AAVrh10.1. mRFP indicates untransduced cells. mGFP indicates Cre-transduced cells.
Violet-colored cells are cells detected with anti-CD31 antibody.
DETAILED DESCRIPTION
This document provides methods and materials for treating a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a PKD).
For example, methods and materials provided herein can be used to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal having, or at risk of developing, a polycystic disease) to treat the mammal. In some cases, nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal can be administered to a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a PKD) to treat the mammal. For example, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be administered to a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a PKD) to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within the mammal (e.g., to treat the mammal). For example, one or more nucleic acid molecules designed to express the components of a targeted gene activation system designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can be administered to a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a P1(D) to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within the mammal (e.g., to treat the mammal).
As used herein, an "increased" level of PC-1 polypeptides and/or PC-2 polypeptides can be any level that is higher than a level of PC-1 polypeptides and/or PC-2 polypeptides in a mammal (e.g., human) that was observed prior to being treated as described herein (e.g., by administering nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides to the mammal). An increase in a level of PC-1 polypeptides and/or PC-2 polypeptides can be in any appropriate tissue and/or organ of a mammal (e.g., a human). Examples of tissues and/or organs in which a level of polypeptides and/or PC-2 polypeptides can be increased as described herein (e.g., by administering nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides to the mammal) include, without limitation, kidneys, liver, spleen, lungs, and brain. In some cases, administering nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides to a mammal having a polycystic disease (e.g., a P1(D) can be effective to increase a level of PC-1 polypeptides and/or PC-2 polypeptides in one or both kidneys in the mammal. For example, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having, or at risk of developing, a polycystic disease such as P1(D) as described herein to increase a level of PC-1 polypeptides and/or PC-2 polypeptides in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having, or at risk of developing, a polycystic disease such as P1(D) as described herein to increase a level of PC-1 polypeptides and/or PC-2 polypeptides in the mammal by, for example, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or more. For example, one or more nucleic acid molecules designed to express the components of a targeted gene activation system designed to activate transcription of a PKD1 gene and/or a PKD2 gene can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having, or at risk of developing, a polycystic disease such as P1(D) as described herein to increase a level of PC-polypeptides and/or PC-2 polypeptides in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In some cases, one or more nucleic acid molecules designed to express the components of a targeted gene activation system designed to activate transcription of a PKD1 gene and/or a PKD2 gene can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having, or at risk of developing, a polycystic disease such as P1(D) as described herein to increase a level of PC-polypeptides and/or PC-2 polypeptides in the mammal by, for example, 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, or more.
In some cases, a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a P1(D) can be treated as described herein (e.g., by administering nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal) to reduce or eliminate one or more symptoms of a polycystic disease (e.g., a P1(D) and/or one or more complications associated with a polycystic disease (e.g., a PKD). Examples of symptoms of a polycystic disease (e.g., a P1(D) and complications associated with a polycystic disease (e.g., a P1(D) include, without limitation, back pain, side pain, headache, a feeling of fullness (e.g., in the abdomen), increased size of the abdomen (e.g., due to an enlarged kidney), blood in the urine, high blood pressure, loss of kidney function (e.g., kidney failure), heart valve abnormalities (e.g., mitral valve prolapse), colon problems (e.g., diverticulosis), development of an aneurysm (e.g., a brain aneurysm), and endothelial dysfunction (ED). For example, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having, or at risk of developing, a P1(D) as described herein to reduce the severity of one or more symptoms of a PDK and/or one or more complications associated with PKD by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, nucleic acid designed to express one or more gene therapy components (or the gene therapy components themselves) designed to activate transcription of a PKD1 gene and/or a PKD2 gene can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having, or at risk of developing, a P1(D) as described herein to reduce the severity of one or more symptoms of a PDK and/or one or more complications associated with PKD by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
In some cases, a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a P1(D) can be treated as described herein (e.g., by administering nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal) to reduce or eliminate one or more cysts (e.g., one or more renal cysts) within the mammal. For example, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having one or more cysts to associate with a polycystic disease such as P1(D) as described herein to reduce the size (e.g., volume) of a cyst within the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, nucleic acid designed to express one or more gene therapy components (or the gene therapy components themselves) designed to activate transcription of a PKD1 gene and/or a PKD2 gene can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having one or more cysts associated with a polycystic disease such as P1(D) as described herein to reduce the cystic index (also referred to as a cystic burden; e.g., the percentage of an organ such as a kidney that is occupied by cysts) in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. Any appropriate method can be used to determine the size of a cyst (e.g., a renal cyst) and/or a cystic index within a mammal (e.g., a mammal having, or at risk of developing, a polycystic disease such as PKD). For example, ultrasound, computed tomography (CT) scanning, magnetic resonance imaging (MRI), and/or histological analysis can be used to determine the size of a cyst (e.g., a renal cyst) and/or a cystic index of a mammal (e.g., a mammal having, or at risk of developing, a polycystic disease such as PKD). In some cases, a cystic index can be determined as described elsewhere (see, e.g., Nieto etal., PLoS One, 11(10):e0163063 (2016)).
In some cases, a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a P1(D) can be treated as described herein (e.g., by administering nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal) to reduce the total kidney volume of one or both kidneys within the mammal and/or to reduce the body weight of the mammal. For example, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having one or more cysts associate with a polycystic disease such as P1(D) as described herein to reduce the total kidney volume of a kidney within the mammal and/or to reduce the body weight of the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, nucleic acid designed to express one or more gene therapy components (or the gene therapy components themselves) designed to activate transcription of a PKD1 gene and/or a PKD2 gene can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having one or more cysts associate with a polycystic disease such as P1(D) as described herein to reduce the total kidney volume of a kidney within the mammal and/or to reduce the body weight of the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. Any appropriate method can be used to determine the total kidney volume of a kidney. For example, ultrasound, CT scanning, and/or MRI can be used to determine the weight of a kidney.
Any appropriate mammal having, or at risk of developing, a polycystic disease (e.g., a P1(D) can be treated as described herein (e.g., by administering nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal). Examples of mammals having, or at risk of developing, a polycystic disease (e.g., a P1(D) that can be treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, rat, hamsters, camels, and llamas. In some cases, a human having, or at risk of developing, a polycystic disease (e.g., a P1(D) can be treated by administering nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide to the human. In some cases, a human having, or at risk of developing, a polycystic disease (e.g., a P1(D) can be treated by administering nucleic acid designed to express one or more gene therapy components (or the gene therapy components themselves) designed to activate transcription of a PKD1 gene and/or a PKD2 gene to the human.
Any appropriate polycystic disease can be treated as described herein (e.g., by administering nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal). Examples of polycystic diseases that can be treated as described herein include, without limitation, PKDs such as ADPKD type 1 and ADPKD
type 2. In some cases, a mammal (e.g., a human) having, or at risk of developing, PKD
(e.g., ADPKD) can be treated by administering nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide to the mammal.
In some cases, a mammal (e.g., a human) having, or at risk of developing, PKD
(e.g., ADPKD) can be treated by administering nucleic acid designed to express one or more gene therapy components (or the gene therapy components themselves) designed to activate transcription of a PKD1 gene and/or a PKD2 gene to the mammal.
When treating a mammal having, or at risk of developing, a polycystic disease (e.g., a P1(D) as described herein (e.g., by administering nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal), the mammal can have one or more cysts present in and/or on any tissue or organ within the mammal.
Examples of tissues and organs within a mammal having a polycystic disease (e.g., a P1(D) that can have one or more cysts include, without limitation, the kidney, the liver, seminal vesicles, pancreas, and arachnoid membrane. For example, a mammal (e.g., a human) having a polycystic disease (e.g., a P1(D) can have one or more renal cysts (e.g., one or more cysts present on or within one or both kidneys).
In some cases, methods for treating a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a P1(D) also can include identifying a mammal as having, or as being at risk of developing, a polycystic disease (e.g., a PKD).
Any appropriate method can be used to identify a mammal as having, or as being at risk of developing, a polycystic disease (e.g., a PKD). For example, imaging techniques (e.g., ultrasound, CT scan, and MRI), laboratory tests (e.g., genetic testing for mutation of one or both copies of the PKD1 gene and/or mutation of one or both copies of the PKD2 gene present in a mammal), and/or generation of family pedigrees can be used to identify a mammal as having, or as being at risk of developing, a polycystic disease (e.g., a PKD).
Once identified as having, or as being at risk of developing, a polycystic disease (e.g., a PKD), the mammal (e.g., the human) can be administered, or instructed to self-administer, nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal as described herein.
In some cases, nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal can include nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide.
Nucleic acid designed to express PC-1 polypeptides and/or PC-2 polypeptides within a mammal can express any appropriate PC-1 polypeptide and/or any appropriate PC-2 polypeptide.

In some cases, the methods and materials provided herein can include administering to a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a P1(D) nucleic acid designed to express a PC-1 polypeptide. Examples of PC-1 polypeptides and nucleic acids encoding PC-1 polypeptides include, without limitation, those set forth in the National Center for Biotechnology Information (NCBI) databases at, for example, accession no. NM 001009944 (version NM 001009944.3), and accession no. AAC34211 (version AAC34211.1).
In some cases, a nucleic acid encoding a PC-1 polypeptide can have an nucleotide sequence set forth in SEQ ID NO:1 (see, e.g., Figure 12A). In some cases, a PC-polypeptide can have an amino acid sequence set forth in SEQ ID NO:2 (see, e.g., Figure 12B).
In some cases, a variant of a PC-1 polypeptide can be used in place of or in addition to a PC-1 polypeptide. A variant of a PC-1 polypeptide can have the amino acid sequence of a naturally-occurring PC-1 polypeptide with one or more (e.g., e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more) amino acid deletions, additions, substitutions, or combinations thereof, provided that the variant retains the function of a naturally-occurring PC-1 polypeptide.
In some cases, the methods and materials provided herein can include administering to a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a P1(D) nucleic acid designed to express a PC-2 polypeptide.
Examples of PC-2 polypeptides and nucleic acids encoding PC-2 polypeptides include, without limitation, those set forth in the National Center for Biotechnology Information (NCBI) databases at, for example, accession no. NR 156488 (version NR 156488.2), and accession no. Q13563 (version Q13563.3).
In some cases, a nucleic acid encoding a PC-2 polypeptide can have an nucleotide sequence set forth in SEQ ID NO:3 (see, e.g., Figure 13A). In some cases, a PC-polypeptide can have an amino acid sequence set forth in SEQ ID NO:4 (see, e.g., Figure 13B).
In some cases, a variant of a PC-2 polypeptide can be used in place of or in addition to a PC-2 polypeptide. A variant of a PC-2 polypeptide can have the amino acid sequence of a naturally-occurring PC-1 polypeptide with one or more (e.g., e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more) amino acid deletions, additions, substitutions, or combinations thereof, provided that the variant retains the function of a naturally-occurring PC-2 polypeptide.
Any appropriate amino acid residue set forth in SEQ ID NO:2 and/or any appropriate amino acid residue set forth in SEQ ID NO:3 can be deleted, and any appropriate amino acid residue (e.g., any of the 20 conventional amino acid residues or any other type of amino acid such as ornithine or citrulline) can be added to or substituted within the sequence set forth in SEQ ID NO:2 and/or SEQ ID NO:4. The majority of naturally occurring amino acids are L-amino acids, and naturally occurring polypeptides are largely comprised of L-amino acids. D-amino acids are the enantiomers of L-amino acids. In some cases, a polypeptide provided herein can contain one or more D-amino acids. In some embodiments, a polypeptide can contain chemical structures such as 6-aminohexanoic acid; hydroxylated amino acids such as 3-hydroxyproline, 4-hydroxyproline, (5R)-5-hydroxy-L-lysine, allo-hydroxylysine, and 5-hydroxy-L-norvaline; or glycosylated amino acids such as amino acids containing monosaccharides (e.g., D-glucose, D-galactose, D-mannose, D-glucosamine, and D-galactosamine) or combinations of monosaccharides.
Amino acid substitutions can be made, in some cases, by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at particular sites, or (c) the bulk of the side chain. For example, naturally occurring residues can be divided into groups based on side-chain properties:
(1) hydrophobic amino acids (norleucine, methionine, alanine, valine, leucine, and isoleucine); (2) neutral hydrophilic amino acids (cysteine, serine, and threonine); (3) acidic amino acids (aspartic acid and glutamic acid); (4) basic amino acids (asparagine, glutamine, histidine, lysine, and arginine); (5) amino acids that influence chain orientation (glycine and proline); and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups can be considered conservative substitutions. Non-limiting examples of substitutions that can be used herein for SEQ ID
NO:2 and/or SEQ ID NO:4 include, without limitation, substitution of valine for alanine, .. lysine for arginine, glutamine for asparagine, glutamic acid for aspartic acid, serine for cysteine, asparagine for glutamine, aspartic acid for glutamic acid, proline for glycine, arginine for histidine, leucine for isoleucine, isoleucine for leucine, arginine for lysine, leucine for methionine, leucine for phenyalanine, glycine for proline, threonine for serine, serine for threonine, tyrosine for tryptophan, phenylalanine for tyrosine, and/or leucine for valine. Further examples of conservative substitutions that can be made at any appropriate position within SEQ ID NO:2 and/or SEQ ID NO:4 are set forth in Table 1 below.
Table 1. Examples of conservative amino acid substitutions.
Original Residue Exemplary substitutions Preferred substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn Asn Glu Asp Asp Gly Pro Pro His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleucine Leu Leu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala Leu Pro Gly Gly Ser Thr Thr Thr Ser Ser Trp Tyr Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Leu, Met, Phe, Ala, Norleucine Leu In some cases, a variant of a PC-1 polypeptide can be designed to include the amino acid sequence set forth in SEQ ID NO:2 with the proviso that it includes one or more non-conservative substitutions. Non-conservative substitutions typically entail exchanging a member of one of the classes described above for a member of another class. Whether an amino acid change results in a functional polypeptide can be determined by assaying the specific activity of the polypeptide using, for example, the methods described herein.
In some cases, a variant of a PC-1 polypeptide having an amino acid sequence with at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99.0%) sequence identity to the amino acid sequence set forth in SEQ
ID NO:2, provided that it includes at least one difference (e.g., at least one amino acid addition, deletion, or substitution) with respect to SEQ ID NO:2, can be used.
In some cases, a variant of a PC-2 polypeptide can be designed to include the amino acid sequence set forth in SEQ ID NO:4 with the proviso that it includes one or more non-conservative substitutions. Non-conservative substitutions typically entail exchanging a member of one of the classes described above for a member of another class. Whether an amino acid change results in a functional polypeptide can be determined by assaying the specific activity of the polypeptide using, for example, the methods described herein.
In some cases, a variant of a PC-2 polypeptide having an amino acid sequence with at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99.0%) sequence identity to the amino acid sequence set forth in SEQ
ID NO:4, provided that it includes at least one difference (e.g., at least one amino acid addition, deletion, or substitution) with respect to SEQ ID NO:4, can be used.
The percent sequence identity between a particular nucleic acid or amino acid sequence and a sequence referenced by a particular sequence identification number (e.g., SEQ ID NO:2 and/or SEQ ID NO:4) is determined as follows. First, a nucleic acid or amino acid sequence is compared to the sequence set forth in a particular sequence identification number using the BLAST 2 Sequences (Bl2seq) program from the stand-alone version of BLASTZ containing BLASTN version 2Ø14 and BLASTP version 2Ø14. This stand-alone version of BLASTZ can be obtained online at fr.com/blast or at ncbi.nlm.nih.gov. Instructions explaining how to use the Bl2seq program can be found in the readme file accompanying BLASTZ. Bl2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. To compare two nucleic acid sequences, the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g., C:\seql.txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g., C: \seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g., C:\output.txt); -q is set to -1; -r is set to 2; and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two sequences: C:\Bl2seq c:\seql.txt -j c:\seq2.txt -p blastn -o c:\output.txt -q -1 -r 2. To compare two amino acid sequences, the options of Bl2seq are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C: \seql.txt); -j is set to a file containing the second amino acid sequence to be compared (e.g., C: \seq2.txt); -p is set to blastp; -o is set to any desired file name (e.g., C:\output.txt);
and all other options are left at their default setting. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\B12seq c: \seql.txt -j c:\seq2.txt -p blastp -o c:\output.txt.
If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
Once aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences. The percent sequence identity is determined by dividing the number of matches by the length of the sequence set forth in the identified sequence (e.g., SEQ ID
NO:2 and/or SEQ ID NO:4), followed by multiplying the resulting value by 100.
It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 75.11, 75.12, 75.13, and 75.14 is rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 is rounded up to 75.2. It also is noted that the length value will always be an integer.
In some cases, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be the form of a vector (e.g., a viral vector or a non-viral vector). In cases where the methods and materials provided herein include nucleic acid designed to express a PC-1 polypeptide and nucleic acid designed to express a PC-2 polypeptide, the nucleic acid designed to express a PC-1 polypeptide and the nucleic acid designed to express a PC-2 polypeptide can be present in the same vector or in separate vectors.
In some cases, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be used for transient expression of a PC-1 polypeptide and/or a PC-2 polypeptide. In some cases, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be used for stable expression of a PC-1 polypeptide and/or a PC-2 polypeptide. In cases where nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide is used for stable expression of a PC-1 polypeptide and/or a PC-2 polypeptide, the nucleic acid encoding a PC-1 polypeptide and/or the nucleic acid encoding a PC-2 polypeptide can be engineered to integrate into the genome of a cell.
Nucleic acid can be engineered to integrate into the genome of a cell using any appropriate method. For example, gene editing techniques (e.g., CRISPR or TALEN
gene editing) can be used to integrate nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide into the genome of a cell.
When a vector used to deliver nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide to a mammal is a viral vector, any appropriate viral vector can be used. A viral vector can be derived from a positive-strand virus or a negative-strand virus. A viral vector can be derived from a virus with a DNA genome or a RNA genome. In some cases, a viral vector can be a chimeric viral vector. In some cases, a viral vector can infect dividing cells. In some cases, a viral vector can infect non-dividing cells. In some cases, a viral vector can be a helper dependent (HD) viral vector. Examples of virus-based vectors that can be used to deliver nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide to a mammal include, without limitation, virus-based vectors based on Ads (e.g., HDAds), AAVs, lentiviruses (LVs), measles viruses, Sendai viruses, herpes viruses, or vesicular stomatitis viruses (VSVs). In some cases, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be delivered to a mammal using a HDAd vector. In some cases, nucleic acid designed to express a PC-2 polypeptide can be delivered to a mammal using an AAV
vector. In some cases, a viral vector including nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can have low seroprevalence in a mammal to be treated as described herein.
When a vector used to deliver nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide to a mammal (e.g., a human) is a non-viral vector, any appropriate non-viral vector can be used. In some cases, a non-viral vector can be an expression plasmid (e.g., a cDNA expression vector).

In some cases, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be administered to a mammal complexed with lipids, polymers, nanoparticles (e.g., nanospheres), and/or lipid nanoparticles (LNPs). For example, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be complexed to one or more LNPs.
In addition to nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide, nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal can contain one or more regulatory elements operably linked to the nucleic acid encoding a PC-1 polypeptide and/or the nucleic acid encoding a PC-2 polypeptide. Such regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that modulate expression (e.g., transcription or translation) of a nucleic acid.
The choice of regulatory element(s) that can be included in a vector depends on several factors, including, without limitation, inducibility, targeting, and the level of expression desired. For example, a promoter can be included in a vector to facilitate transcription of a nucleic acid encoding a PC-1 polypeptide and/or nucleic acid encoding a PC-2 polypeptide. A promoter can be a naturally occurring promoter or a recombinant promoter. A promoter can be ubiquitous or inducible (e.g., in the presence of tetracycline), and can affect the expression of a nucleic acid encoding a polypeptide in a general or tissue-specific manner (e.g.,a cadherin 16 (Cdh16 or Ksp-cadherin) promoter sequence such as a mouse Cdh16 promoter sequence). Examples of promoters that can be used to drive expression of a PC-1 polypeptide and/or PC-2 polypeptide include, without limitation, EFla promoter sequences, CBh promoter sequences, PKD1 promoter sequences, PKD2 promoter sequences, cytomegalovirus (CMV) promoter sequences (e.g., human CMV promoter sequences), Rous sarcoma virus (RSV) promoter sequences, aquaporin 2 (AQP2) promoter sequences, gamma-glutamyltransferase 1 (Ggtl) promoter sequences, and Ksp-cadherin promoter sequences. As used herein, "operably linked"
refers to positioning of a regulatory element in a vector relative to a nucleic acid encoding a polypeptide in such a way as to permit or facilitate expression of the encoded polypeptide. For example, a vector can contain a promoter and nucleic acid encoding a PC-1 polypeptide. In this case, the promoter is operably linked to a nucleic acid encoding a PC-1 polypeptide such that it drives expression of the PC-1 polypeptide in cells. In cases where a vector contains both nucleic acid designed to express a PC-1 polypeptide and nucleic acid designed to express a PC-2 polypeptide, the nucleic acid designed to express a PC-1 polypeptide and the nucleic acid designed to express a PC-2 polypeptide can be operably linked to the same promoter or different promoters.
In some cases, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can contain nucleic acid encoding a detectable label. For example, a vector can include nucleic acid designed to express a PC-1 polypeptide and nucleic acid encoding a detectable label positioned such that the encoded polypeptide is a fusion polypeptide that includes a PC-1 polypeptide fused to a detectable polypeptide. In some cases, a detectable label can be a peptide tag. Examples of detectable labels that can be used as described herein include, without limitation, HA
tags, Myc-tags, FLAG-tags, fluorescent polypeptides (e.g., green fluorescent polypeptides (GFPs), and mCherry polypeptides), luciferase polypeptides, and sodium iodide symporter (NIS) polypeptides.
Nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be produced by techniques including, without limitation, common molecular cloning, polymerase chain reaction (PCR), chemical nucleic acid synthesis techniques, and combinations of such techniques. For example, PCR or RT-PCR can be used with oligonucleotide primers designed to amplify nucleic acid (e.g., genomic DNA or RNA) encoding a PC-1 polypeptide or a PC-2 polypeptide.
In some cases, a vector including nucleic acid designed to express a PC-1 polypeptide can be a HDAd vector including nucleic acid designed to express a polypeptide that is operably linked to a CBh promoter sequence. An exemplary HDAd vector including nucleic acid encoding a PC-1 polypeptide that is operably linked to a CBh promoter sequence can include the nucleic acid sequence set forth in SEQ
ID NO:5.
In some cases, a vector including nucleic acid designed to express a PC-2 polypeptide can be a AAV vector including nucleic acid designed to express a polypeptide that is operably linked to a EFla promoter sequence. An exemplary AAV
vector including nucleic acid encoding a PC-2 polypeptide that is operably linked to a EFla promoter sequence can include the nucleic acid sequence set forth in SEQ
ID NO:6.

In some cases, a vector including nucleic acid designed to express a PC-1 polypeptide can be a HDAd vector including nucleic acid designed to express a polypeptide that is operably linked to a CBh promoter sequence and include nucleic acid designed to express a PC-2 polypeptide that is operably linked to a EFla promoter sequence. An exemplary HDAd vector including nucleic acid encoding a PC-1 polypeptide that is operably linked to a CBh promoter sequence and including nucleic acid encoding a PC-2 polypeptide that is operably linked to a EFla promoter sequence can include the nucleic acid sequence set forth in SEQ ID NO:7.
In some cases, nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal can include one or more nucleic acid molecules designed to express gene therapy components designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides). For example, nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal can include one or more nucleic acid molecules designed to express the components of a targeted gene activation system (e.g., designed for CRISPR-Cas9-based targeted gene activation system) designed to upregulate transcription of the PKD1 gene and/or the PKD2 gene to increase the level of PC-1 polypeptides and/or PC-2 polypeptides in cells. Any appropriate targeted gene activation system can be used (e.g., a synergistic activation mediators (SAM) system). In some cases, a targeted gene activation system can include (a) a fusion polypeptide including a deactivated Cos (dCas) polypeptide and a transcriptional activator polypeptide, (b) one or more helper activator polypeptides, and (c) a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the one or more helper activator polypeptides. For example, nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal can include (a) nucleic acid that can express a fusion polypeptide including a deactivated Cas (dCas) polypeptide and a transcriptional activator polypeptide, (b) nucleic acid that can express one or more helper activator polypeptides, and (c) nucleic acid that can express a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the one or more helper activator polypeptides.
A fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide in a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can include any appropriate dCas polypeptide.
Examples of dCas polypeptides that can be included in a fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide that can be used as a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene can include, without limitation, deactivated Cas9 (dCas9) polypeptides (e.g., deactivated Streptococcus pyogenes Cas9 (dSpCas9), deactivated Staphylococcus aureus Cas9 (dSaCas9), and deactivated Campylobacter jejuni Cas9 (dCjCas9)), and deactivated Cas phi (dCasO) polypeptides.
.. In some cases, a dCas polypeptide that can be included in a fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide that can be used as a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene can be as described elsewhere (see, e.g., Konermann et al., Nature, Jan 29;517(7536):583-8 (2015) at, for example, the Supplementary Materials; Sajwan et al., Sci Rep., 9:18104 (2019) at, for example, Supplementary Materials; Jiang et al., Biosci. Rep., 39(8):BSR20191496 (2019) at, for example, Table 1). A dCas polypeptide in a fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide in a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can be encoded by any appropriate nucleic acid sequence.
A fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide in a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can include any appropriate transcriptional activator polypeptide. In some cases, a transcriptional activator polypeptide can recruit an RNA polymerase. In some cases, a transcriptional activator polypeptide can recruit one or more transcription factors and/or transcription co-factors (e.g., RNA
polymerase co-factors). Examples of transcriptional activator polypeptides that can be included in a fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide that can be used in a targeted gene activation system (e.g., a SAM
system) designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene can include, without limitation, polypeptides having four copies of viral protein 16 (VP64 polypeptides). In some cases, a transcriptional activator polypeptide that can be included in a fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide that can be used in a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene can be as described elsewhere (see, e.g., Konermann et al., Nature, Jan 29;517(7536):583-8 (2015) at, for example, the Supplementary Materials;
Sajwan et al., Sci Rep., 9:18104 (2019) at, for example, Supplementary Materials; Jiang et al., Biosci. Rep., 39(8):B5R20191496 (2019) at, for example, Table 1). A
transcriptional activator polypeptide in a fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide in a targeted gene activation system (e.g., a SAM
system) designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can be encoded by any appropriate nucleic acid sequence.
A fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide in a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can include the dCas polypeptide and the transcriptional activator polypeptide in any orientation. In some cases, a transcriptional activator polypeptide can be fused to the N-terminus of a dCas polypeptide. In some cases, a transcriptional activator polypeptide can be fused to the C-terminus of a dCas polypeptide.

In some cases, a fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide in a targeted gene activation system (e.g., a SAM
system) designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can include a dSpCas9 polypeptide and a VP64 polypeptide. For example, a fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide that can be used in a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can be a dCas9-VP64 fusion polypeptide.
A fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide in a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can be encoded by any appropriate nucleic acid sequence.
A targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can include any appropriate helper activator polypeptide.
Examples of helper activator polypeptides that can be used in a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene can include, without limitation, Escherichia virus M52 coat protein (M52) polypeptides, nuclear factor NF-kappa-B p65 subunit (p65) polypeptides, heat shock factor protein 1 (HSF1) polypeptides, VP64 polypeptides. In some cases, a helper activator polypeptide can include two or more (e.g., two, three, or more) helper activator polypeptides. For example, a helper activator polypeptide can be a fusion polypeptide including two or more helper activator polypeptides. For example, a helper activator polypeptide can be a complex including two or more helper activator polypeptide. In some cases, a helper activator polypeptide can include a M52 polypeptide, a p65 polypeptide, and a HSF1 polypeptide (a M52-P65-HSF1 (MPH) polypeptide). In some cases, a helper activator polypeptide that can be used in a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene can be as described elsewhere (see, e.g., Konermann et al., Nature, Jan 29;517(7536):583-8 (2015) at, for example, the Supplementary Materials; Sajwan et al., Sci Rep., 9:18104 (2019) at, for example, Supplementary Materials; Jiang et al., Biosci. Rep., 39(8):B5R20191496 (2019) at, for example, Table 1). A helper activator polypeptide in a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can be encoded by any appropriate nucleic acid sequence.
A targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can include any appropriate nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the helper activator polypeptide. A nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene can be any appropriate length. In some cases, a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene can include from 19 nucleotides to 21 nucleotides.
In some cases, a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the helper activator polypeptide that can be used in a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene can include a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene. A
nucleic acid sequence that is complementary to a target sequence within a PKD1 gene can include any appropriate nucleic acid sequence. A nucleic acid sequence that is complementary to a target sequence within a PKD1 gene can be complementary to (e.g., can be designed to target) any target sequence within a PKD1 gene (e.g., can target any location within a PKD1 gene). In some cases, a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene can be a single stranded nucleic acid sequence. In some cases, a target sequence within a PKD1 gene can be in a promoter sequence of the PKD1 gene. In some cases, a target sequence within a PKD1 gene can be from about 1 nucleotide to about 200nuc1eotides away from a promoter sequence of the PKD1 gene.
Examples of nucleic acid sequences that are complementary to a target sequence within a PKD1 gene include, without limitation, nucleic acid sequences that can be encoded by a nucleic acid sequence including the sequence TCGCGCTGTGGCGAAGGGGG (SEQ ID
NO:13), a nucleic acid sequence including the sequence CCAGTCCCTCATCGCTGGCC
(SEQ ID NO:14), and a nucleic acid sequence including the sequence GGAGCGGAGGGTGAAGCCTC (SEQ ID NO:15).
In some cases, a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the helper activator polypeptide that can be used in a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene can include a nucleic acid sequence that is complementary to a target sequence within a PKD2 gene. A
nucleic acid sequence that is complementary to a target sequence within a PKD2 gene can include any appropriate nucleic acid sequence. A nucleic acid sequence that is complementary to a target sequence within a PKD2 gene can be complementary to (e.g., can be designed to target) any target sequence within a PKD2 gene (e.g., can target any location within a PKD2 gene). In some cases, a nucleic acid sequence that is complementary to a target sequence within a PKD2 gene can be a single stranded nucleic acid sequence. In some cases, a target sequence within a PKD2 gene can be in a promoter sequence of the PKD2 gene. In some cases, a target sequence within a PKD2 gene can be from about 1 nucleotide to about 200 nucleotides away from a promoter sequence of the PKD2 gene.
Examples of nucleic acid sequences that are complementary to a target sequence within a PKD2 gene include, without limitation, nucleic acid sequences that can be encoded by a nucleic acid sequence including the sequence ACGCGGACTCGGGAGCCGCC (SEQ ID
NO:23), a nucleic acid sequence including the sequence ATCCGCCGCGGCGCGCTGAG (SEQ ID NO:24), and a nucleic acid sequence including the sequence GTGCGAGGGAGCCGCCCCCG (SEQ ID NO:25).

A nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene that can be included in a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the helper activator polypeptide in a targeted gene activation system (e.g., a SAM
system) designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can be encoded by any appropriate nucleic acid sequence. In some cases, nucleic acid sequences that encode a nucleic acid that is complementary to a target sequence within a PKD1 gene can be encoded by a nucleic acid sequence shown in Table 2 or Table 3.
In some cases, a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the helper activator polypeptide that can be used in a targeted gene activation system (e.g., a SAM system) designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene can include any appropriate nucleic acid sequence that can bind the helper activator polypeptide. In some cases, a nucleic acid sequence that can bind the helper activator polypeptide can bind a M52 polypeptide. Examples of nucleic acid sequences that can bind the helper activator polypeptide (e.g., a M52 polypeptide) can include, without limitation, nucleic acid sequences that can be encoded by a nucleic acid sequence including the sequence ACATGAGGATCACCCATGT (SEQ ID NO:26). A nucleic acid sequence that can bind the helper activator polypeptide that can be included in a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the helper activator polypeptide in a targeted gene activation system (e.g., a SAM
system) designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can be encoded by any appropriate nucleic acid sequence.
In addition to nucleic acid designed to express one or more gene therapy components designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides), nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal can contain one or more regulatory elements operably linked to nucleic acid that can express (a) a fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide, (b) nucleic acid that can express one or more helper activator polypeptides, and/or (c) nucleic acid that can express a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the one or more helper activator polypeptides. Such regulatory elements can include promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that modulate expression (e.g., transcription or translation) of a nucleic acid. The choice of regulatory element(s) that can be included in a vector depends on several factors, including, without limitation, inducibility, targeting, and the level of expression desired. For example, a promoter can be included in a vector to facilitate transcription of a nucleic acid that can express (a) a fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide, (b) a nucleic acid that can express one or more helper activator polypeptides, and/or (c) a nucleic acid that can express a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the one or more helper activator polypeptides. A promoter can be a naturally occurring promoter or a recombinant promoter. A promoter can be ubiquitous or inducible (e.g., in the presence of tetracycline), and can affect the expression of a nucleic acid encoding a polypeptide in a general or tissue-specific manner (e.g., AQP2 promoter sequences, Ggtl promoter sequences, and Ksp-cadherin promoter sequences). Examples of promoters that can be used to drive expression of (a) a fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide, (b) one or more helper activator polypeptides, and/or (c) a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the one or more helper activator polypeptides include, without limitation, EFla promoter sequences, CBh promoter sequences, CMV
promoter sequences (e.g., human CMV promoter sequences), RSV promoter sequences, U6 promoter sequences, AQP2 promoter sequences, Ggtl promoter sequences, and Ksp-cadherin promoter sequences. As used herein, "operably linked" refers to positioning of a regulatory element in a vector relative to a nucleic acid encoding a polypeptide or a nucleic acid (e.g., an RNA) in such a way as to permit or facilitate expression of the encoded polypeptide or the transcribed nucleic acid. For example, a vector can contain a promoter and nucleic acid encoding a fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide. In this case, the promoter is operably linked to a nucleic acid encoding a fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide such that it drives expression of the fusion polypeptide including a dCas polypeptide and a transcriptional activator polypeptide in cells. In cases where a vector contains both a nucleic acid that can express one or more helper activator polypeptides and a nucleic acid that can express a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the one or more helper activator polypeptides, the nucleic acid that can express one or more helper activator polypeptides and the nucleic acid that can express a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the one or more helper activator polypeptides can be operably linked to the same promoter or different promoters. In cases where a vector contains each of a nucleic acid that can express (a) a fusion polypeptide including dCas polypeptide and a transcriptional activator polypeptide, (b) a nucleic acid that can express one or more helper activator polypeptides, and (c) a nucleic acid that can express a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the one or more helper activator polypeptides, the nucleic acid that can express the fusion polypeptide including dCas polypeptide and a transcriptional activator polypeptide, the nucleic acid that can express the nucleic acid that can express one or more helper activator polypeptides, and the nucleic acid that can express a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene and/or a PKD2 gene, and (ii) a nucleic acid sequence that can bind the one or more helper activator polypeptides can be operably linked to the same promoter or different promoters. In cases where two or more nucleic acid sequences are operably linked to a single promoter, the coding sequences of each nucleic acid sequence can be separated by a sequence encoding a cleavage signal (e.g., P2A cleavage signal).
In some cases, one or more nucleic acid molecules designed to express the components of a targeted gene activation system designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can be the form of one or more vectors (e.g., viral vectors and/or non-viral vectors). In some cases, one or more nucleic acid molecules designed to express the components of a targeted gene activation system designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene can be present in the same vector or in separate vectors.
When a vector used to deliver one or more nucleic acid molecules designed to express the components of a targeted gene activation system designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene to a mammal is a viral vector, any appropriate viral vector can be used. A viral vector can be derived from a positive-strand virus or a negative-strand virus. A viral vector can be derived from a virus with a DNA genome or a RNA genome. In some cases, a viral vector can be a chimeric viral vector. In some cases, a viral vector can infect dividing cells. In some cases, a viral vector can infect non-dividing cells. Examples virus-based vectors that can be used to deliver nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide to a mammal include, without limitation, virus-based vectors based on Ads (e.g., HDAds), AAVs, LVs, measles viruses, Sendai viruses, herpes viruses, or VSVs.
When a vector used to deliver one or more nucleic acid molecules designed to express the components of a targeted gene activation system designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene to a mammal (e.g., a human) is a non-viral vector, any appropriate non-viral vector can be .. used. In some cases, a non-viral vector can be an expression plasmid (e.g., a cDNA
expression vector).

In some cases, one or more nucleic acid molecules designed to express the components of a targeted gene activation system designed to activate transcription of a PKD1 gene and/or to activate transcription of a PKD2 gene can be administered to a mammal by direct injection of nucleic acid molecules complexed with lipids, polymers, nanoparticles (e.g., nanospheres), and/or LNPs. For example, nucleic acid designed to express a PC-1 polypeptide and/or nucleic acid designed to express a PC-2 polypeptide can be complexed to one or more LNPs.
In some cases, one or more nucleic acid molecules designed to express the components of a targeted gene activation system designed to activate transcription of a .. PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can be in a HDAd vector (e.g., in a single HDAd vector) including (a) nucleic acid encoding a dCas9VP64 fusion polypeptide that is operably linked to a CMV
promoter sequence, (b) nucleic acid encoding a MPH polypeptide that is operably linked to a EFla promoter sequence, and (c) nucleic acid encoding a nucleic acid molecule (e.g., gRNA) including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene, and (ii) a nucleic acid sequence that can bind a MS2 polypeptide that is operably linked to a U6 promoter sequence. Exemplary HDAd vectors including (a) nucleic acid encoding a dCas9VP64 fusion polypeptide that is operably linked to a CMV
promoter sequence, (b) nucleic acid encoding a MPH polypeptide that is operably linked to a EFla promoter sequence, and (c) nucleic acid encoding a nucleic acid molecule (e.g., gRNA) including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene, and (ii) a nucleic acid sequence that can bind a MS2 polypeptide that is operably linked to a U6 promoter sequence can include, without limitation, the nucleic acid sequence set forth in SEQ ID NO:8, and the nucleic acid sequence set forth in SEQ ID NO:9.
In some cases, one or more nucleic acid molecules designed to express the components of a targeted gene activation system designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can be in the form of two or more AAV vectors including (a) nucleic acid encoding a dCas9VP64 fusion polypeptide that is operably linked to a EFla promoter sequence, (b) nucleic acid encoding a MPH polypeptide that is operably linked to a CMV
promoter sequence, and (c) nucleic acid encoding a nucleic acid molecule (e.g., gRNA) including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene, and (ii) a nucleic acid sequence that can bind a MS2 polypeptide that is operably linked to a U6 promoter sequence. For example, a first AAV vector can include (a) nucleic acid encoding a dCas9VP64 fusion polypeptide that is operably linked to a EFla promoter sequence, and a second AAV vector can include (b) nucleic acid encoding a MPH polypeptide that is operably linked to a CMV promoter sequence, and (c) nucleic acid encoding a nucleic acid molecule (e.g., gRNA) including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene, and (ii) a nucleic acid sequence that can bind a MS2 polypeptide that is operably linked to a U6 promoter sequence. An exemplary AAV vector including (a) nucleic acid encoding a dCas9VP64 fusion polypeptide that is operably linked to a EFla promoter sequence can include the nucleic acid sequence set forth in SEQ ID NO:10. An exemplary AAV vector including (b) nucleic acid encoding a MPH polypeptide that is operably linked to a CMV
promoter sequence, and (c) nucleic acid encoding a nucleic acid molecule (e.g., gRNA) including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene, and (ii) a nucleic acid sequence that can bind a M52 polypeptide that is operably linked to a U6 promoter sequence can include the nucleic acid sequence set forth in SEQ
ID NO:11.
In some cases, one or more nucleic acid molecules designed to express the components of a targeted gene activation system designed to activate transcription of a PKD1 gene (e.g., resulting in an increased level of PC-1 polypeptides) and/or to activate transcription of a PKD2 gene (e.g., resulting in an increased level of PC-2 polypeptides) can be in the form of an AAV vector (e.g., a single AAV vector) including (a) nucleic acid encoding a dCas01 polypeptide that is operably linked to a CBh promoter sequence, (b) nucleic acid encoding a MPH polypeptide that is operably linked to the CBh promoter sequence, and (c) nucleic acid encoding a nucleic acid molecule (e.g., gRNA) including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene, and (ii) a nucleic acid sequence that can bind a M52 polypeptide that is operably linked to a U6 promoter sequence. An exemplary AAV vector including (a) nucleic acid encoding a dCas01 polypeptide that is operably linked to a CBh promoter sequence, (b) nucleic acid encoding a MPH polypeptide that is operably linked to the CBh promoter sequence, and (c) nucleic acid encoding a nucleic acid molecule (e.g., gRNA) including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene, and (ii) a nucleic acid sequence that can bind a MS2 polypeptide that is operably linked to a U6 promoter sequence can include the nucleic acid sequence set forth in SEQ
ID NO:12.
Any appropriate method can be used to deliver nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal to a mammal (e.g., a human). For example, nucleic acid designed to increase a level of PC-polypeptides and/or PC-2 polypeptides within a mammal can be administered locally or systemically. For example, nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal can be administered locally by retro-ureter injection and/or subcapsular injection to a mammal (e.g., a human). For example, nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal can be administered systemically by i.p. injection and/or i.v.
injection to a mammal (e.g., a human).
Also provided herein are methods for improving delivery of nucleic acid (e.g., vectors such as viral vectors) to a mammal (e.g., to one or more cells within a mammal).
For example, inducing proteinuria in a mammal prior to administering nucleic acid can be effective to improve delivery of nucleic acid to one or more cells (e.g., from blood within a mammal into one or more cells) within a mammal. In some cases, a mammal can first be administered one or more LPSs (e.g., to induce proteinuria in the mammal), and can subsequently be administered nucleic acid. For example, a mammal having, or at risk of developing, a polycystic disease (e.g., PKD) can first be administered one or more LPSs, and can subsequently be administered nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within the mammal (e.g., to improve delivery of nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides to one or more cells within a mammal).
Any appropriate LPS having the ability to induce proteinuria in a mammal (e.g., a human) can be used to improve delivery of nucleic acid to cells within the mammal as described herein. In some cases, another agent (e.g., an agent that is not an LPS) that can induce proteinuria in a mammal (e.g., a human) can be used in place of or in addition to one or more LPSs to improve delivery of nucleic acid to a mammal (e.g., to one or more cells within a mammal). An agent that can induce proteinuria in a mammal can be any type of molecule (e.g., a polypeptide, and a small molecule). In some cases, an agent that can induce proteinuria in a mammal can be a cell-opening agent. Examples of agents that can induce proteinuria and be used as described herein include, without limitation, puromycin, adriamycin, protamine sulfate, cationic albumin, and polycations.
In some cases, administering one or more LPSs (and/or another agent or agents that can induce proteinuria in a mammal) prior to administering nucleic acid can be effective to improve delivery of the nucleic acid to the mammal (e.g., to one or more cells within a mammal) by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent (e.g., as compared to the amount of nucleic acid delivered to a mammal that has not been administered one or more LPSs and/or other agent(s) that can induce proteinuria in a mammal).
In some cases, administering one or more LPSs (and/or another agent or agents that can induce proteinuria in a mammal) prior to administering nucleic acid can be effective to deliver large nucleic acid to the mammal (e.g., to one or more cells within a mammal). For example, administering one or more LPSs (and/or another agent or agents that can induce proteinuria in a mammal) prior to administering nucleic acid can be effective to deliver nucleic acid having a size of from about 0.15 kb to about 36 kb (e.g., from about 0.15 kb to about 33 kb, from about 0.15 kb to about 30 kb, from about 0.15 kb to about 28 kb, from about 0.15 kb to about 25 kb, from about 0.15 kb to about 20 kb, from about 0.15 kb to about 17 kb, from about 0.15 kb to about 15 kb, from about 0.15 kb to about 12 kb, from about 0.15 kb to about 10 kb, from about 0.15 kb to about 8 k1:0 from about 0.15 kb to about 5 k1:0 from about 0.15 kb to about 3 kb, from about 0.15 kb to about 1 k1:0 from about 0.15 kb to about 0.5 k1:0 from about 0.5 kb to about 36 kb, from about 1 kb to about 36 k1:0 from about 5 kb to about 36 kb, from about 8 kb to about 36 kb, from about 10 kb to about 36 kb, from about 15 kb to about 36 kb, from about 20 kb to about 36 kb, from about 25 kb to about 36 kb, from about 30 kb to about 36 kb, from about 0.5 kb to about 30 kb, from about 1 kb to about 25 k1:0 from about 5 kb to about 20 k1:0 from about 10 kb to about 15 k1:0 from about 1 kb to about 5 kb, from about 5 kb to about 10 kb, from about 15 kb to about 20 kb, from about 20 kb to about 25 kb, from about 25 kb to about 30 kb, or from about 30 kb to about 35 kb) to the mammal (e.g., to one or more cells within a mammal). For example, administering one or more LPSs (and/or another agent or agents that can induce proteinuria in a mammal) prior to administering nucleic acid can be effective to deliver nucleic acid having a mass of from about 10 kilodaltons (kDa) to about 50 kDa (e.g., from about 10 kDa to about 50 kDa, from about 10 kDa to about 40 kDa, from about 10 kDa to about 30 kDa, from about 10 kDa .. to about 20 kDa, from about 20 kDa to about 40 kDa, from about 25 kDa to about 35 kDa, from about 15 kDa to about 20 kDa, from about 20 kDa to about 25 kDa, from about 25 kDa to about 30 kDa, from about 30 kDa to about 35 kDa, from about 35 kDa to about 40 kDa, from about 40 kDa to about 45 kDa, or from about 45 kDa to about 50 kDa) to the mammal (e.g., to one or more cells within a mammal). For example, administering one or more LPSs .. (and/or another agent or agents that can induce proteinuria in a mammal) prior to administering nucleic acid can be effective to deliver nucleic acid having a diameter of from about 10 nm to about 26 nm (e.g., from about 10 nm to about 25 nm, from about

10 nm to about 20 nm, from about 10 nm to about 17 nm, from about 10 nm to about 15 nm, from about 10 nm to about 12 nm, from about 12 nm to about 26 nm, from about 15 nm to about 26 nm, from about 18 nm to about 26 nm, from about 20 nm to about 26 nm, from about 22 nm to about 26 nm, from about 12 nm to about 20 nm, from about 15 nm to about 18 nm, from about 12 nm to about 15 nm from about 18 nm to about 20 nm, or from about 20 nm to about 22 nm) to the mammal (e.g., to one or more cells within a mammal).
Any appropriate amount of one or more LPSs (and/or another agent or agents that can .. induce proteinuria in a mammal) can be administered to a mammal (e.g., a human) to improve delivery of nucleic acid to any type of cell within the mammal. For example, from about 7 milligrams per kilogram body weight (mg/kg) to about 9 mg/kg of one or more LPSs (and/or another agent or agents that can induce proteinuria in a mammal) can be administered to a mammal (e.g., a human) to improve delivery of nucleic acid to any type of cell within the mammal.
One or more LPSs (and/or another agent or agents that can induce proteinuria in a mammal) can improve delivery of nucleic acid to any type of cell within a mammal.
Examples of types of cells that an agent that can induce proteinuria in a mammal can improve delivery of nucleic acid to include, without limitation, kidney cells (e.g., renal tubule epithelial cells and/or proximal tubule cells such as proximal tubule cells adjacent to glomeruli), spleen cells, lungs cells, and brain cells.

One or more LPSs (and/or another agent or agents that can induce proteinuria in a mammal) can be administered to a mammal (e.g., a human) at any appropriate time before nucleic acid is administered to the mammal. In some cases, one or more LPSs (and/or another agent or agents that can induce proteinuria in a mammal) can be administered to a mammal (e.g., a human) at least 18 hours prior to administering nucleic acid to the mammal. For example, one or more LPSs (and/or another agent or agents that can induce proteinuria in a mammal) can be administered to a mammal (e.g., a human) from about 18 hours to about 24 hours prior to administering nucleic acid to the mammal.
Any appropriate method can be used to deliver one or more LPSs (and/or another agent or agents that can induce proteinuria in a mammal) to a mammal (e.g., a human).
For example, one or more LPSs (and/or another agent or agents that can induce proteinuria in a mammal) can be administered locally or systemically. For example, one or more LPSs (and/or another agent or agents that can induce proteinuria in a mammal) can be administered locally by retro-ureter injection and/or subcapsular injection to a mammal (e.g., a human). For example, one or more LPSs (and/or another agent or agents that can induce proteinuria in a mammal) can be administered systemically by i.p.
injection and/or i.v. injection to a mammal (e.g., a human).
In some cases, methods for treating a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a PKD) can include administering to the mammal nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal as the sole active ingredient to treat the mammal.
In some cases, methods for treating a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a PKD) as described herein (e.g., by administering nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal) also can include administering to the mammal one or more (e.g., one, two, three, four, five or more) additional active agents (e.g., therapeutic agents) that are effective to treat one or more symptoms of a PKD and/or one or more complications associated with a polycystic disease (e.g., a PKD) to treat the mammal.
Examples of additional active agents that can be used as described herein to treat one or more .. symptoms of a polycystic disease (e.g., a PKD) and/or one or more complications associated with a polycystic disease (e.g., a PKD) include, without limitation, an inhibitor of a vasopressin receptor (e.g., tolvaptan), angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARBs), pain relievers (e.g., acetaminophen), antibiotics, pasireotide, and anti-miR-17 oligonucleotide RGLS4326. In some cases, the one or more additional active agents can be administered together with the administration of the nucleic acid designed to increase a level of PC-1 polypeptides and/or polypeptides within a mammal. For example, a composition containing nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal also can include one or more additional active agents that are effective to treat one or more symptoms of a polycystic disease (e.g., a PKD) and/or one or more complications associated with a polycystic disease (e.g., a PKD). In some cases, the one to or more additional active agents that are effective to treat one or more symptoms of a polycystic disease (e.g., a PKD) and/or one or more complications associated with a polycystic disease (e.g., a PKD) can be administered independent of the administration of the nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal. When the one or more additional active agents that are effective to treat one or more symptoms of a polycystic disease (e.g., a PKD) and/or one or more complications associated with a polycystic disease (e.g., a PKD) are administered independent of the administration of the nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal, the nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal can be administered first, and the one or more additional active agents that are effective to treat one or more symptoms of a polycystic disease (e.g., a PKD) and/or one or more complications associated with a polycystic disease (e.g., a PKD) performed second, or vice versa.
In some cases, methods for treating a mammal (e.g., a human) having, or at risk of developing, a polycystic disease (e.g., a PKD) as described herein (e.g., by administering nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal) also can include subjecting the mammal one or more (e.g., one, two, three, four, five or more) additional treatments (e.g., therapeutic interventions) that are effective to treat one or more symptoms of a polycystic disease (e.g., a PKD) and/or one or more complications associated with a polycystic disease (e.g., a PKD) to treat the mammal. Examples of additional treatments that can be used as described herein to treat one or more symptoms of a polycystic disease (e.g., a PKD) and/or one or more complications associated with a polycystic disease (e.g., a P1(D) include, without limitation, consuming a restricted diet (e.g., a diet low in methionine, high in choline, and/or high in betaine content), maintaining a healthy body weight, exercising regularly, undergoing dialysis, undergoing a kidney transplant, and dietary ketosis. In some cases, the one or more additional treatments that are effective to treat one or more symptoms of a polycystic disease (e.g., a P1(D) and/or one or more complications associated with a polycystic disease (e.g., a P1(D) can be performed at the same time as the administration of the nucleic acid designed to increase a level of PC-1 polypeptides and/or polypeptides within a mammal. In some cases, the one or more additional treatments that are effective to treat one or more symptoms of a polycystic disease (e.g., a P1(D) and/or one or more complications associated with a polycystic disease (e.g., a P1(D) can be performed before and/or after the administration of the nucleic acid designed to increase a level of PC-1 polypeptides and/or PC-2 polypeptides within a mammal.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1: Expression of PC-1 Polypeptides and/or PC-2 Polypeptides to Treat ADPKD
This Example describes vectors that can be used as genetic therapies for treating ADPKD by delivering the cDNA of the PKD1 gene, the cDNA of the PKD2 gene, or both (e.g., simultaneously). Both viral and non-viral delivery methods are described.
Results A helper-dependent adenoviral vector that expresses PKD1, PKD2, or both HDAds with all the Ad genome viral open reading frames removed has space for genetic cargo up to 35 kb. AAVs can deliver the 2.9 kb PKD2 cDNA while HDAds can deliver the 12.9 kb PKD1 cDNA or a combination of the PKD1 and PKD2 cDNAs.

Materials and Methods HDAd Vectors HD-Ad PKD1 vectors were generated that contained a PKD1 cDNA. GFP-Luciferase HDAd vectors were also generated for transduction testing.
A helper virus was used to provide the missing Ad genes and proteins for HDAd vectors. If a normal Ad was used as the helper virus, both the helper and the HDAd virus was packaged, producing a preparation that was contaminated by the helper virus. To avoid this contamination problem, the Ad helper virus has its packaging signal flanked by two LoxP sites.
When the HDAd vector and LoxP-modified helper virus are delivered into 116 cells that overexpress the Cre recombinase, Cre excises the helper virus' packaging signal, blocking its packaging, and significantly reducing helper virus contamination.
This system routinely produces yields of HDAd of 1013 virus particles (vp) with helper virus contamination below 0.02%.
HDAd was passaged up to 6 times and then purified on 2 CsC1 gradients. Once purified, each virus preparation was sequenced to verify identity, and the amount of vector and helper virus was measured by qPCR.
Testing HDAd Vectors Once produced, vectors are tested in vitro in 293 and RCTE human cells and IMCD mouse cells. The cells are infected at varied multiplicities of infection (MOD of each vector. GFP fluorescence are analyzed by fluorescence microscopy and cell lysates will be prepared at the peak time of expression (usually day 2). Once GFPLuc expression is validated for each of the vectors, the vectors proceed to in vivo testing in RC mice.
Groups of 5 male and 5 female mice are injected with each of the vectors by the retro-.. ureter route and sub-capsular routes. One group of male and female mice is injected with PBS as negative controls. Luciferase imaging is performed under isoflurane anesthesia on day 1 and 7. After luciferase imaging, all of the mice are euthanized using CO2. Both kidneys are sectioned to identify the cells that are expressing GFP using antibodies against GFP and EpCAM as well as staining with biotinylated lotus tetragonolobus lectin (LTL) to label mature proximal tubules and papillary collecting ducts. The percent transgene protein positive tubule cells are quantified using ImageJ based on pixel counts.

The level of gene delivery in the renal pelvis, distal and proximal tubule, and in the glomerulus are determined. ANOVA comparisons are used to compare injection methods and promoters.
Each vector is used to transduce PKD1 and PKD2 null mutant cells and PC-1 and PC-2 expression by the vectors is verified by western blot.
Shorter Term In Vivo Therapeutic Testing The vectors are injected into 1 month old RC/RC mice that are early in the PKD

disease process. Each virus for injection is blinded. Mice are injected in the right kidney by the retro-ureter route in groups of 10 male and 10 female mice with PBS, HDAd-GFPLuc, HDAd-PKD1, or HDAd-PKD1 and PKD2. Cyst status for mice is established by MRI. The kidneys of the mice are monitored by MRI imaging bi-weekly to assess if vector injection into the right kidney delays cystogenesis progression relative to the uninjected kidneys. Serum creatinine and BUN are measured at varied times to assess kidney function.
Five animals from each group are sacrificed at one week and five animals from each group are sacrificed at one month. Luciferase imaging is performed in the GFP-Luciferase groups just prior to sacrifice to document the persistence of expression mediated by the HDAd vectors. The injected right kidney and the uninjected left kidney are weighed to determine kidney mass to body mass ratios. One half of each kidney is used for western blot and qPCR to determine whether PKD1 expression and PC-1 protein levels are increased. The remaining half is sectioned to identify the cells that are expressing exogenous human PC-1 and for histological examination to examine effects on cyst index, number and growth. Sections are stained by H&E to monitor changes in cyst sizes and infiltration of immune cells into the tissue.
HDAd-PKD/ or HDAd-PKD/ and PKD2 therapies can mediate changes in kidney size and cystic phenotypes relative to control vector and to PBS-injected controls.
It is also examined if combined PKD1 and PKD2 provides better balanced expression than PKD1 alone.
Longer Term In Vivo Therapeutic Testing The Shorter Term testing described above is repeated, but over longer times with larger group sizes. Five animals from each group are sacrificed at one month, five animals from each group are sacrificed 3 months, five animals from each group are sacrificed 6 months, and five animals from each group are sacrificed at 9 months.
Luciferase imaging is performed and gene expression, kidney size, creatinine, BUN, kidney mass, and cyst formation is evaluated to determine if HDAd-PKD/ therapy .. mediates changes in kidney size and cystic phenotypes relative to control vector and to PBS-injected controls and uninjected kidneys.
Example 2: Targeted Gene Activation to Treat ADPKD
This Example describes gene activation machinery capable of increasing expression of the wild type PKD1 gene.
Results Targeted gene activation of the PKD1 allele in human 293 (adrenal-derived) cells Three separate lentiviral vectors were produced, each of which expressed one of the three components of the Cas9-SAM system and a different selectable marker.
Human 293 cells were transduced with the first lentivirus to express dCas9VP64 and selected for with blasticidin. Subsequently, cells were transduced with the second lentivirus to express MPH and selected for with hygromycin. Lastly, cells were transduced with the third lentivirus to express an sgRNA targeting the human PKD1 promoter and selected for with zeocin (Figure 2).
After this process produced a stable bulk population of modified 293 cells, RNA
was purified from the cells. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify the relative levels ofPKD1 mRNA in the transduced cells versus untransduced cells (Figure 3). Expression of human sgRNA1 brought mRNA to a relative level of 7.9, human sgRNA2 brought it to 13.8, and human sgRNA3 brought it to 3.1. Therefore, each of these sgRNA's were effective at increasing the level ofPKD1 mRNA, and also at different levels.
Targeted gene activation of the PKD1 allele in human renal cortical tubule epithelial (RCTE) cells Human RCTE cells were subjected to the same process described above through the qRT-PCR step (Figure 4). Expression of human sgRNA1 brought PKD1 mRNA to a .. relative level of 2.9, human sgRNA2 brought it to 9.7, and human sgRNA3 brought it to 1.7. The order of activation strength of these sgRNA's was conserved between 293 and RCTE cells, indicating that targeting particular promoter sequences may hold more inherent activation strength regardless of cell type.
Targeted gene activation of the Pkdl allele in mouse inner medullary collecting duct (1114CD3) cells Mouse IMCD3 cells were subjected to the same process described above through the qRT-PCR step, with the exception that expressed sgRNA's were targeted to sequences in the mouse Pkdl promoter rather than the human PKD1 promoter (Figure 5).
Expression of mouse sgRNA1 brought Pkdl mRNA to a relative level of 2.8, mouse sgRNA2 brought it to 51.5, mouse sgRNA3 brought it to 8.4, and mouse sgRNA5 brought it to 5.1. In this case, a control sgRNA targeted to the promoter of the mouse Rib gene was used as a control, which elevated the Pkdl transcript to a level of 2.8, possibly due to dysregulation of cellular transcriptional networks.
Molecular cloning of dual AAV vector SAM plasmids and verification of protein expression and sgRNA sequences After sgRNAs compatible with activation of the human PKD1 and mouse Pkdl genes were identified, construction of vectors for in vivo delivery of Cas9-SAM
components began. One of these vectors is a HDAd capable of carrying all three components of the SAM system (Figure 6A). Although less commonly used in vivo, a second option is a lentiviral vector carrying all components of the same system (Figure 6B). The three components of the SAM system are too large to be packaged into a single AAV vector, so a third option is a dual AAV vector system, where the first AAV
delivers MPH and the sgRNA and the second AAV delivers dCas9VP64 (Figure 6C). While the Cas9-SAM system described thus far is too large to be packaged into a single AAV
vector, the newly discovered Casill protein is small enough to make single AAV
vector amenable to delivering Cas01, MPH, and an sgRNA (Figure 6D).
The first component of the SAM system, dCas9VP64, is 4.4 kb in length, which is already large for AAV. To ensure successful packaging, the transgene was flanked by relatively small expression elements in the AAV construct (Figure 6C). To ensure robust dCas9VP64 expression from these expression cassettes, the vector production plasmids were transfected into 293 cells and dCas9VP64 protein was assayed three days later via western blot (Figure 7). dCas9VP64 was detected in three different AAV
expression cassettes with different combinations of promoters and polyadenylation signals as well as an adenoviral expression cassette. The lentiviral expression cassette transfected did not produce detectable dCas9VP64 protein. This assay confirmed that the first of two AAV's necessary for the dual vector system is expressing dCas9VP64. The second AAV, which must express MPH and an sgRNA, has been cloned to express one of three human sgRNAs or one of seven mouse Pkdl sgRNA's and sequence verified (Tables 2 and 3).

Table 2. sgRNA sequences used to target the human PKD1 promoter.

t..) Gene Transcript TSS Nucleic acid encoding the o t..) t..) Name ID sgRNA ID Distance Guide Sequence SEQ ID NO
Sequencing result SEQ ID NO 1-vi PKD1 NM 000296 Genscript3 84 TCGCGCTGTGGCGAAGGGGG 13 TCGCGCTGTGGCGAAGGGGG 13 vi .6.
PKD1 NM 000296 Genscriptl 107 CCAGTCCCTCATCGCTGGCC 14 CCAGTCCCTCATCGCTGGCC 14 vi PKD1 NM 000296 Genscript2 133 GGAGCGGAGGGTGAAGCCTC 15 Table 3. sgRNA sequences used to target the mouse Pkdl promoter.
Gene Transcript TSS Nucleic acid encoding the Name ID sgRNA ID Distance Guide Sequence SEQ ID NO
Sequencing result SEQ ID NO
P
mouse Pkdl o Pkdl NM 013630 13 GAAGAGGGCGGAGCCTGTGA 16 SAM sgRNA1 .

, cal , t.) mouse Pkdl Pkdl NM 013630 34 TTGCAGATCCTGCAGTAGGC 17 TTGCAGATCCTGCAGTAGGC 17 " .
SAM sgRNA2 , .
mouse Pkdl , , Pkdl NM 013630 60 TGAAGGAAGGGCGCCCTCAG 18 TGAAGGAAGGGCGCCCTCAG 18 , , SAM sgRNA3 mouse Pkdl Pkdl NM 013630 86 CGCCCAGTGAGCGTGAGCCT 19 SAM sgRNA4 mouse Pkdl Pkdl NM 013630 107 GGTGGGCGGGGTCTCACGGG 20 SAM sgRNA5 mouse Pkdl Pkdl NM 013630 138 GCAGAAGGCGGGGCCTCCGG 21 GCAGAAGGCGGGGCCTCCGG 21 Iv SAM sgRNA6 n 1-i mouse Pkdl Pkdl NM 013630 160 CGCTGGGTCTGCTGCAGACC 22 CGCTGGGTCTGCTGCAGACC 22 cp SAM sgRNA7 t..) o t..) t..) -a-, ,-, t..) .6.
c, ,-, Non-viral delivery of genetic therapies for ADPKD
The same plasmids used for production of the viral vectors described above are complexed with lipid nanoparticles (LNPs) as a lower biosafety risk alternative to viral vectors. This plasmid DNA-LNP complexes is administered intravenously to transfect cells in vivo.
Materials and Methods Generate and Test AA V, Lentiviral, and HDAd Vectors for TGA
A HDAd, a lentiviral vector, and two AAV vectors have been designed to carry the SAM system. Briefly, each expression cassette of dCas9-VP65; MS2-P65-HSF1; and the sgRNA cassette is amplified with oligonucleotides bearing large I-SceI or I-CeuI restriction sites. These products are inserted into unique I-SceI and I-CeuI restriction sites in the HDAd vector pDelta18, pAAV-SceCeu, and pLenti-SceCeu. dCas9-VP64 is amplified with I-SceI
and I-CeuI sites, MS2-P65-HSF1 with I-SceI, and the mouse sgRNA cassettes with ICeuI.
One AAV-dCas9-VP64 is used with three different AAVs expressing MS2-P65-HSF1 and one the one of three mouse sgRNAs. Similarly, there are three HDAds and three different lentiviruses carrying three mouse sgRNAs.
In Vivo Transduction and Therapeutic Testing of Pkdl-TGA Vectors Groups of 10 male and 10 female RC/RC mice are injected with PBS, HDAd-SAM
(as a single vector), Lenti-SAM (as a single vector), or AAV-SAM (as a dual vector system).
Retro-ureter or sub-capsular injection are used. 1011 of HDAd-TGA gRNA vector is injected.
106 transducing units (TU) of VSVg-pseudotyped lentivector with the entire SAM
system is injected. AAV-Pkdl-TGA vectors can mediate therapy, even when they require co-infection of the cell by 2 vectors. AAVrh10 is used robustness and ability to transduce cells with high multiplicity. To maximize co-infection of the same renal cells with 2 AAVs, 1012 vg of both AAVrh10-Pkdl-TGA vectors are delivered to the mice.
RC/RC mice are injected as described above. Each virus sample is blinded. MRI
imaging, serum creatinine, and BUN are measured to assess kidney function.
Five animals from each group are sacrificed at one week and five animals from each group are sacrificed at one month for western blot, qPCR, and histochemistry to determine whether Pkdl expression and PC-1 protein levels are increased in the injected kidney and if there are positive or negative effects on cyst index, number and growth. Sections are stained by H&E
to monitor changes in cyst sizes and immune infiltrates. Gene expression, kidney size, creatinine, BUN, kidney mass, and cyst formation are evaluated to determine if the HDAd, AAV, or lentivirus vectors mediate changes in kidney size and cystic phenotypes relative to controls.
Example 3: Increasing Vector Penetration into Tissues From the Blood Viral or non-viral gene therapy and cancer therapies use vectors that are many megaDaltons in size. These agents have a hard time entering into certain tissues like the kidney and brain after intravenous (i.v.) injections.
This Example describes methods that can loosen intracellular attachments to allow i.v. injected large vectors to penetrate into tissues such as the brain, lungs, spleen, liver, and kidney. For example, lipopolysaccharide (LPS) can be used to promote proteinurea and to increase leak of large vectors from the blood into tissues.
Results Induced proteinuria increases gene delivery to renal tubule epithelial cells Following intravenous administration of Ad or AAV, the vector appears to rarely penetrate past the glomerulus and further into the tubule of the nephron. The filtration properties of the glomerular barrier typically excludes solute in the blood that is greater than 10 kilodaltons (kDa) in mass or 10 nm in diameter. Ad and AAV are both significantly above these thresholds in size and thus are not generally expected to transduce renal tubule epithelial cells after intravenous injection. To overcome this limitation, proteinuria was induced in mice via effacement of podocyte foot processes in the glomerulus, which has been shown to structurally disrupt the glomerular filter and allow larger solute from the blood into the tubule of the nephron.

Luciferase/red-green hybrid reporter mice were intraperitoneally (i.p.) injected with 200 1.tg of lipopolysaccharides (LPS) to induce proteinuria. The next day, mice were given an intravenous injection of PBS, AAV8, AAV9, or AAVrh10 (n=1). In the case of AAV8, the mouse that had been administered LPS showed increase luminescence in its kidneys versus the PBS control (Figure 8). When sectioning the kidneys of these mice, the LPS-injected mouse had consistently transduced (EGFP+) proximal tubules cell adjacent to glomeruli, while the PBS-injected mouse only had transduced cells in its glomeruli (Figure 9).
To quantify the extent to which tubule epithelial cells were being transduced during proteinuria, a larger scale experiment was performed using a lower dose of AAV
(2e11 genome copies per mouse). Mice were injected with either PBS or LPS i.p., and were then injected with AAV8 the following day (n=3 mice for each group) or PBS as control (n=1 mouse for each group). Mice were sacrificed six days after AAV administration and tissues were imaged for luminescence ex vivo. Livers did not show a significant difference in luminescence between PBS and LPS-treated mice (Figure 10A). However, ex vivo kidney luminescence showed a significance increase in LPS-treated mice versus PBS-treated mice (Figure 10B). These kidneys were then homogenized and analyzed by flow cytometry. The cells were first gated into a CD45- population, as to remove hematopoietic cells from the query. The EpCAM+CD31- population, where EpCAM is a marker of epithelial cells and CD31 is a marker of endothelial cells, was then examined. In this population, the percentage of EGFP+ cells in LPS-treated mice was significantly increased from PBS-treated mice, indicating that induced proteinuria was transducing more epithelial cells (Figure 10C).
Transduced endothelial cells were also examined by analyzing the EpCAM-CD31+
population of cells and it was found that there was no significant difference between PBS and LPS-treated mice, indicating that induced proteinuria increased transduction of epithelial but not endothelial cells in the kidney (Figure 10D). The ability to consistently target proximal tubule cells for transduction is useful for being able to treat ADPKD as well as other genetic kidney diseases.
Since AAV showed promising results in renal tubule transduction when combined with induced proteinuria, it was investigated if the same effect could be achieved with a larger Ad vector. Mice were administered PBS or LPS followed by 1" viral particles of Ad5. Kidneys were imaged for luminescence ex vivo and some evidence of increased transduction in the LPS-treated mouse kidneys was observed (Figure 11A). When the signals from these kidneys were quantified, it was found that the kidney luminescence had significantly increased in LPS-treated from PBS-treated (Figure 11B). When livers and kidneys from these mice were sectioned for fluorescent histology, increased transduction was seen in the kidneys of LPS-injected mice, but only in the glomeruli (Figure 11C). The LPS-treated mouse had reduced transduction in the liver compared to PBS-treated mice, possibly due to LPS interaction with the Kupffer cells in the liver.
1E::1 Materials and Methods Animals Mice used in these experiments were Fl hybrids of loxP-STOP-loxP-Luciferase (LSL-Luc) mice (The Jackson Laboratory Stock No: 005125) and membrane-tomato/membrane-green (mT/mG) mice (The Jackson Laboratory Stock No: 007676).
Thus, each mouse endogenously expressed tdTomato, and upon Cre-recombinase expression in a particular cell, has activated luciferase and EGFP genes.
Proteinuria induction in mice Urine was collected from mice of various ages and a baseline level of proteinuria was determined using Beyer Albustix. Mice were then injected with 200 [ig of LPS
(dissolved at 1 mg/mL in otherwise sterile PBS) intraperitoneally. Approximately 24 hours later, urine was collected and proteinuria levels were again determined. In most cases, administration of LPS versus a PBS control clearly caused an increased level of proteinuria in mice.
Viral vector delivery After induction of proteinuria via administration of LPS or a PBS control, mice were injected with adeno-associated virus serotype 8 (AAV8) expressing Cre recombinase or replication-defective adenovirus serotype 5 (RDAd5) expressing Cre recombinase intravenously via tail vein injection. Injection volumes were 100 [EL. The dose of AAV8-Cre administered ranged from 2e11 to 1.94e12 genome copies while the dose of RDAd5-Cre administered was 1 ell viral particles.
Luminescent imaging After viral vector injection, luminescent signals were monitored and quantified in vivo in mice until the signal peaked (observed to be six days) using Perkin Elmer IVIS
Lumina and Living Image software. To do this, mice were anesthetized with isoflurane and injected intraperitoneally with luciferin, and imaged 10 minutes later. At the six day time point, mice were sacrificed and their tissues were dissected and placed in a six well plate to be imaged ex vivo and these signals were quantified. In some cases, the kidneys were laterally bisected to enhance the luminescent signal being emitted from within the tissue.
Fluorescent histology The same tissues used for luminescent imaging were processed for fluorescent histology. Kidneys and liver were fixed in 4% paraformaldehyde overnight and then soaked in 15% sucrose/PBS followed by 30% sucrose/PBS until the tissues sank. Tissues were frozen in blocks in Optimal Cutting Temperature (OCT) medium. A Leica cryostat was used to section tissues at a thickness of 18 [tM and mount them on glass slides.
Mounting Medium with DAPI (Vector Labs) was then dropped on the sections and a glass coverslip was placed on top of the slide. Confocal microscopy was performed using a Zeiss LSM780 microscope with optimized settings to image tdTomato, EGFP, and DAPI.
Flow cytometry Kidney samples were chopped into small pieces using scissors and put in Miltenyi tubes. 2.35 mL of DMEM was added. 100 pL of enzyme D, 50 pL of enzyme R, and 12.5 pL of enzyme A from the Miltenyi "Tumor Dissociation Kit" into were added to each sample. Program 37C mTDK 1 or soft tissue dissociation was used on the OctoMACS
machine. C-Tube was washed well by pouring DMEM, inverting, and passing through a 70 [tM filter (15 mL volume). Cells were then spun at 400 xg for 10 minutes.
Samples were resuspended into 3.1 mL of cold DPBS and 900 [EL of Miltenyi Debris removal solution was added and resuspended well. 4 mL of ice cold DPBS was carefully overlayed onto the samples. Samples were spun at 3000 g for 10 minutes with brakes on. 1 mL of ACK Lysis buffer was added for 1 minute and subsequently quenched by filling the tube to top (15 mL
rol) with cold RPMI. All samples were processed and passed through filters and transferred to 5 mL flow tubes. Tubes were filled with PBS and spun at 400g for 5 minutes.
500 [IL of MasterMix was added to each sample to stain for flow cytometry, as follows:
EpCAM
PECy7 (1:250) (BioLegend, Cat# 118216), CD31 AF647 32 (1:500) (BioLegend, Cat#

102516), CD45 perCP (1:1000) (BioLegend, Cat# 103130), Viability ¨ ghost dye red 780 (1:2000) (Tonbo Biosciences, Cat# 13-0865-T100), FC block (1:500) (BD
Pharmingen, Cat#
553141). Results were analyzed using FlowJo software.
Example 4: Induced Proteinuria Enhances Adeno-Associated Virus Transduction of Renal Tubule Epithelial Cells after Intravenous Administration There are a variety of genetic diseases of the kidney tubule that might be amenable to correction via gene therapy. However, gene delivery to renal tubule epithelial cells mediated by viral vectors via the blood is historically inefficient due to the permselectivity of the glomerular barrier, which typically will not allow molecules larger than 50 kilodaltons in mass or 10 nanometers in diameter to pass into the tubule of the nephron.
This Example demonstrates that AAV vectors can penetrate into the nephron and transduce tubule epithelial cells in a state of proteinuria.
Results .. AA V8 gene delivery to the kidney is distinctly enhanced in a state of induced proteinuria To begin to investigate the effects of induced proteinuria on viral vector gene delivery to the kidney, mice were administered an i.p. injection of 200 pg of LPS. The mode of delivery and dose were as described elsewhere (Reiser et al., I Clin. Invest., 113:1390-1397 (2004)). The following morning, urine was collected from mice injected with either LPS or PBS as a control and assayed using a proteinuria dipstick to ascertain whether proteinuria had effectively been induced (example portrayed in Figure 22). Subsequently, mice were administered i.v. injections of self-complementary AAV8-Cre (scAAV8-Cre), scAAV9-Cre, scAAVrh10-Cre, or PBS as control (n = 1 for each combination of PBS or LPS and each vector). The mice used in this experiment are known as LSL-Luc-mT/mG Fl hybrid mice:
each mouse has one LoxP-STOP-LoxP-Luciferase allele and one membrane-targeted tdTomato/membrane-targeted EGFP allele at the ROSA locus. Thus, each mouse has luciferase and mG genes activatable by Cre-expressing vectors, allowing for tracking of vector pharmacodynamics on both a cellular and tissue-specific level (Figure 14A).
Luciferase activity in the mice was tracked daily via bioluminescent imaging until the signals reached an approximate plateau at day 6 (Figure 23A). The signals measured in vivo almost were almost certainly emitted from luciferase activity in the livers of these, due to the high liver tropism of the three AAV serotypes used (Figure 14B). To directly assess liver and kidney transduction of the injected mice, the mice were sacrificed and these organs were imaged ex vivo. While kidneys of the AAV9 and AAVrh10 injected mice with or without induced proteinuria exhibited minimal luminescence which was localized to the renal pelvis region of the kidney, the kidneys of the mouse with induced proteinuria injected with AAV8 had pervasive luciferase expression throughout the entire kidney (Figure 14B).
This observation of increased luciferase expression through the whole of the kidney tissue while in a state of proteinuria, as opposed to the luciferase activity seen exclusively on the edges of the kidney capsule of the control mouse, indicates a clear difference in vector pharmacodynamics between mice in states of induced proteinuria and not.
To assess kidney transduction on a cell-by-cell basis, the kidney and liver tissues were sectioned to view direct fluorescence via confocal microscopy. In the current reporter mouse model system, untransduced cells will endogenously express membrane-targeted tdTomato (mT), while Cre-expressing transduced cells will stop expressing tdTomato and begin to express membrane-targeted EGFP (mG). For each of the three AAV
serotypes, it was observed that treating mice with LPS prior to AAV injection resulted in many instances of transduced cells with tubular morphology adjacent to glomeruli, as compared to control kidneys (Figure 15). To determine if viral vectors might bypass the glomerulus and penetrate the most proximal part of the nephron, the proximal tubule, and to verify which additional cells AAV is transducing in an induced proteinuria state, kidney sections were counterstained with lotus tetragonolobus lectin (LTL), a marker of proximal tubule cells. No instances of EGFP + transduced cells seemed to be double positive for the LTL stain. This indicates that although induced proteinuria seems to allow AAV to penetrate further into kidney tissue from the blood and transduce more tubule cells, these cells are not necessarily proximal tubule cells.
AAV8 significantly increases renal epithelial cell transduction during proteinuria Data indicate that AAV serotypes 8, 9, and rh10 each potentially increase transduction of renal tubule epithelial cells when mice are in an induced state of proteinuria.
In particular, AAV8 had the most striking effect in terms of increased transduction during induced proteinuria (Figure 14B). To quantify this effect, and to determine if this effect could be achieved at a lower dose, new groups of mice were given an i.p.
administration of .. either PBS or LPS at Day -1 and an i.v. administration of scAAV8-Cre at Day Oat a dose of 2ell genome copies (GC). Proteinuria dipsticks from these groups of mice at Day -1 (baseline) and Day 0 (post PBS or LPS) are shown as an example (Figure 22).
These mice were imaged for in vivo luminescence at Day 6 at which point the mice were sacrificed and their tissues imaged ex vivo. There was no significant difference observed between PBS and LPS-injected groups in vivo (indicative of liver transduction), liver ex vivo, or brain ex vivo (Figure 16A). Although insignificant, brain luminescence was increased in all samples, indicating that LPS administration may induce some blood brain barrier disruption and increase transduction of cells in the brain. In contrast to the livers and brains, ex vivo kidney transduction visibly increased in LPS-injected mice versus PBS-injected mice (Figure 16B). Upon quantitation of luminescence in these kidneys, the kidneys of the LPS-injected mice exhibited significantly higher luminescence than those of the PBS-injected mice (Figure 16C). These kidneys were then processed for flow cytometry and labeled to detect epithelial cell adhesion molecule (EpCAM), a marker of epithelial cells, CD31, a marker of endothelial cells, and various other immune cell markers.
Upon examination of the %EGFP+ (transduced) cells in EpCAM+ CD31- and EpCAM- CD31+
populations, it was found that epithelial cells, but not endothelial cells, had a significant increase in transduction, indicating that the injected AAV8 did in fact have more access to epithelial cells during a state of induced proteinuria (Figure 16C). In addition, an increased, albeit insignificant, %EGFP+ macrophages were found in the blood of LPS-injected mice as compared to PBS-injected mice, indicating that an increased presence of macrophages may have been induced by LPS administration and subsequently transduced by scAAV8-Cre (Figure 24B). Representative flow plots and gating strategies are shown in Figure 25.
AAVrh10 significantly increases hematopoietic cell, but not epithelial cell transduction, during LPS-induced proteinuria It was next sought to determine if a particular serotype of AAV could in fact result in a significantly increased number of epithelial cells in the kidney after i.v.
injection in a state of induced proteinuria. In the initial experiment, AAV8 had stronger results than AAV9 or AAVrh10. To ascertain whether particular serotypes of AAV other than AAV8 might be able transduce significantly more renal epithelial cells in a state of induced proteinuria, the prior flow cytometry experiment was repeated using scAAVrh1O-Cre rather than scAAV8-Cre. The %EGFP+ (transduced) present among CD45- (non-hematopoietic) and CD45+

(hematopoietic) cells in the kidneys was examined (Figure 17A). There was no difference in transduced CD45- cells between PBS and LPS-injected groups. However, there was a .. significant increase in transduced CD45 + cells. This effect may be due to an increased number of hematopoietic cells that infiltrated the kidney after LPS injection and were more susceptible to transduction the day after.
The transduction of epithelial cells in the kidney was examined. As with the previous experiment using scAAV8, the %EGFP+ cells amongst CD45- EpCAM+ and CD45- CD31+
populations, which represent transduced epithelial cells and transduced endothelial cells, respectively, was examined. When this experiment was performed using scAAV8 (Figure 16), there was a significant increase in transduced epithelial cells, but not endothelial cells, between the LPS and PBS-injected mice. However, when this experiment was repeated using scAAVrh10, there was no significant difference between the LPS and PBS-injected groups of mice (Figure 17B). To further drill down on proximal tubule cells, a specific subset of kidney tubule epithelial cells, samples were also labeled with LTL
and aquaporin-1 (AQP1). In both cases, no significant difference was observed between transduced cells in LPS or PBS-injected mice. Overall, mice with or without induced proteinuria did not seem to have a change in transduced renal epithelial cells after intravenous injection of scAAVrh1O-Cre. Representative flow plots and gating strategies are shown in Supplemental Figure 18.
A naturally liver-detargeted vector enhances kidney transduction during induced proteinuria Although increasing transduction in tubule cells in the kidney is an important goal for efficacy of gene therapy, detargeting vectors from off-target tissues is an important facet of gene therapy safety. While AAV8 showed efficacy in terms of increasing kidney transduction during a state of induced proteinuria, it also fully transduces the liver (Figure 24A). To attempt to resolve the off-target tissue transduction, AAV1, a serotype known to have lower liver tropism than other serotypes, was tested in conjunction with induced proteinuria.
Mice were administered an i.p. injection of either PBS or LPS at Day -1 and an i.v.
injection of scAAV1-Cre at Day Oat a dose of 9.95e10 GC. Similar to previous experiments, in vivo luminescence signals peaked at Day 6, at which point mice were sacrificed and ex vivo liver luminescence was comparable between both groups of mice (Figure 18A, top).
Although mean kidney ex vivo luminescence was increased in LPS-injected mice versus PBS-injected mice, the difference was not significant. When comparing this data side-by-side with the ex vivo kidney luminescence data of the scAAV8-Cre injected mice from Figure 16, both PBS-injected and LPS-injected groups of scAAV1-injected mice had higher signals than the LPS and scAAV8-injected mice, and at approximately half of the dose of scAAV8, indicating that scAAV1 may have a higher native kidney tropism both with and without induced proteinuria (Figure 18A, lower). Upon sectioning of the kidneys of these mice to examine endogenous mT and mG fluorescence, mice injected with PBS followed by scAAV1 had many instances of transduced glomerular cells while mice injected with LPS
followed by scAAV1 had increased instances of transduced tubular cells (Figure 18B).
Importantly, the livers of the mice injected with scAAV1 were only partially transduced, while the livers of the mice injected with scAAV8 were fully transduced (Figure 28). These data indicate that scAAV1 may be an ideal vector for targeting renal tubule epithelial cells while avoiding unnecessary transduction of hepatocytes.

Induced proteinuria enhances Ad5 transduction of glomerular, but not epithelial cells Thus far, four different serotypes of AAV were tested in tandem with the LPS-induced proteinuria method. Between these serotypes, notable differences in the transduction profiles of kidney and liver cells were observed. The variation in transduction profiles is likely due to differences in receptor usage as well as capsid surface electromagnetic charges.
To test other applications and potential limitations of the induced proteinuria method with respect to kidney transduction, physically larger gene delivery vector, replication-defective adenovirus serotype 5 expressing Cre recombinase (Ad5-Cre), was used.
Mice were administered i.p. injections of either PBS or LPS on Day -1 and i.v.
injections of Ad5-Cre on Day 0. In vivo luminescent signals (indicative of level of liver transduction) were monitored up to Day 5 until they peaked. In contrast to previous experiments using AAV, mice injected with LPS prior to Ad5-Cre had significantly reduced in vivo luminescence compared to PBS-injected mice (Figure 19A, left). The mice were then sacrificed and their kidneys were imaged for ex vivo luminescence. As with the previous experiments performed with AAV, kidneys from mice administered LPS prior to Ad5-Cre had a significantly higher signal than those from mice administered PBS prior to Ad5-Cre (Figure 19A, right). In the images of the kidneys of PBS and Ad5-Cre injected mice, little to no luminescence is visible, whereas in the kidneys of the LPS and Ad5-Cre injected mice, two out of three of the kidneys showed enhanced luminescence localized near the renal pelvis (Figure 19B). This is in contrast to kidney images of mice injected with LPS
and scAAV8, which showed more diffuse luminescence throughout the kidney (Figure 16).
Kidneys from the Ad5-Cre injected mice were then sectioned to examine endogenous mT and mG fluorescence. Notably, in contrast to previous experiments using AAV, no instances of transduced tubule cells were observed in kidneys of PBS or LPS
and Ad5-Cre injected mice. However, there were observed to be an increased number of glomerular cells transduced in the LPS and Ad5-Cre injected mice versus the PBS-injected mice, indicating that induced proteinuria did not enhance penetration of Ad5-Cre into renal epithelial tubular cells but may have aided penetration further into the glomerulus itself (Figure 19C). In accordance with the in vivo luminescence signals from these mice, liver sectioning showed that mice injected with PBS followed by Ad5-Cre had fully transduced livers while mice injected with LPS followed by Ad5-Cre had only partially transduced livers, possibly as a result of LPS interactions with Kupffer cells (Figure 27A). These data indicate that while the induced proteinuria method used in tandem with Ad may not necessarily be effective in treating genetic diseases of the tubule, such as polycystic kidney disease, it may be helpful in increasing gene delivery to the glomerulus.
Induced proteinuria increases epithelial cell transduction in a mouse model of ADPKD
Thus far, out of a handful of gene therapy vectors tested, only particular vectors tended to increase transduction of renal tubule epithelial cells while mice were in a state of induced proteinuria: namely, AAV1 and AAV8. To test if the induced proteinuria method is amenable to enhancing renal tubule epithelial cell transduction in a mouse model of relevant human disease, this technique was employed on mice with ADPKD. Pkol/RctRc mice, which are homozygous for the hypomorphic Pkdl allele p.R3277C and develop progressive ADPKD similar to human disease, were backcrossed to mT/mG mice until pups had exactly two Pkdrc alleles and at least one mT/mG allele. In essence, the newly generated mice are identical (give or take differences in genetic background due to a partial backcross) to the original mT/mG mice except they now develop ADPKD (Figure 20A).
The Pkdrcv-Rc-mT/mG hybrid mice were administered i.p. injections of PBS or LPS
on Day -1 and an i.v. injection of scAAV8-Cre at Day 0 at a dose of 2e11 GC.
Under the assumption that vector pharmacodynamics would recapitulate those of the prior AAV
experiments, mice were sacrificed at Day 6 and their tissues were sectioned.
While evidence of glomerular transduction was apparent in the mouse injected with PBS
followed by scAAV8-Cre, evidence of tubular cell transduction was observed only in the mouse injected with LPS followed by scAAV8-Cre (Figure 20B). The livers of these mice were fully transduced by scAAV8-Cre, as expected (Figure 29). Overall, these data support mice with ADPKD being amenable to increased tubule cell transduction and thereby enhanced potential for gene therapy by the induced proteinuria method.

Materials and Methods Animal studies All experiments were carried out according to the provisions of the Animal Welfare Act, PHS Animal Welfare Policy, the principles of the NIH Guide for the Care and Use of Laboratory Animals.
AAV vectors AAV vectors were produced using a standard triple transfection and iodixanol gradient purification method. Briefly, a vector plasmid (pTRS-CBh-Cre), a rep and cap plasmid (pRC), and a pHelper plasmid were transfected into 293T cells using polyethylenimine. Three days later, cells were harvested and lysed by successive freeze/thaw cycles. Cell lysate was overlayed onto an iodixanol gradient and ultracentrifuged for two hours. The banded AAV was extracted via needle and syringe and titrated via qPCR using SYBRTM Green. All AAV vectors used in this study were self-complementary (scAAV) with a cytomegalovirus chicken 13-actin hybrid promoter (CBh) driving expression of the Cre recombinase gene.
Ad vectors Replication-defective Ad vectors were produced in 293 cells and were purified by double banding on CsC1 gradients. Cre expression is driven by the CMV
promoter.
Flow cytometry Kidney samples were chopped into small pieces using scissors and put in Miltenyi tubes. 2.35 mL of Gibco DMEM (cat # 11054001), 100 [EL of enzyme D, 50 [EL of enzyme R, and 12.5 [EL of enzyme A from the Miltenyi "Tumor Dissociation Kit" were added into each sample. Samples were homogenized using soft tissue dissociation program on Miltenyi OctoMACSTm Separator. Samples were passed through 70 [EM filters and spun at 400 x g for 10 minutes. Pellets were resuspended in 3.1 mL of cold DPBS, treated with 900 [EL of Miltenyi Debris removal solution, overlayed with 4 mL of ice cold DPBS, and spun at 3000 x g for 10 minutes. The samples were washed with DPBS and red blood cells were lysed with 1 mL of ACK Lysis buffer for 1 minute. The samples were resuspended in 900 [EL
of RPMI
and filtered using 35 [EM flow tube filters.
Fluorescent staining occurred as follows: After all samples were processed and passed through filters, they were washed twice with PBS. Samples were stained with a master mix composed of EpCAM PECy7 (1:250) (BioLegend, Cat# 118216), CD31 (1:500) (BioLegend, Cat# 102516), CD45 perCP (1:1000) (BioLegend, Cat#
103130), TCRf3 BV421 (1:1000), CD4 BV510 32 [EL (1:500), CD8 BV570 (1:500), CD1lb BV650 (1:1000), Ghost Dye Red 780 (1:2000) (Tonbo Biosciences, Cat# 13-0865-T100), and FC
block (1:500) (BD Pharmingen, Cat# 553141). Three minutes prior to experimental mice being sacrificed, 3 [Eg of CD45 BV711 was injected intravenously to be able to distinguish between circulating and tissue resident CD45 + cells. Samples were stained for 30 minutes at 4C in the dark, washed twice with PBS and ran on CytekTM Aurora spectral flow cytometer.
For the experiments also staining against a-Fucose, Lotus Tetragonolobus Lectin (LTL), Biotinylated (1:100) (Vector Laboratories, Cat# B-1325-2) was the primary stain and BV786 Streptavidin (1:2000) (BD Horizon, Cat# 563858) was the secondary stain.
For the experiments also staining against Aquaporin-1, Anti-Aquaporin-1 (1:100) (Boster Biological Technology, Cat# PB9473) was the primary stain and anti-rabbit AF647 (1:2000) (Invitrogen, Cat# A-21245) was the secondary stain. In these experiments, CD31 was stained using anti-CD31 BV510 (1:150) (BD Biosciences, Cat# 740124).
In vivo bioluminescent imaging Mice were anesthetized with isoflurane and injected intraperitoneally with 150 [EL of D-Luciferin (20 mg/mL; RR Labs Inc., San Diego, CA). Images were taken using PerkinElmer IVIS Lumina S5 Imaging System ten minutes after D-Luciferin administration and luminescence was quantified using Living Image software. During ex vivo tissue imaging, tissues were placed in either 6-well or 12-well tissue culture vessels and imaged. In all cases except for Figure 14, kidneys were laterally bisected before imaging to prevent squelching of luminescence by the kidney capsule.

Statistical Analyses All statistical analyses were performed using GraphPad Prism 9. p-values were generated using Mann-Whitney tests unless otherwise noted.
Tissue sectioning and confocal microscopy Tissues from mice with membrane-bound fluorescent proteins were fixed by overnight immersion in 4% paraformaldehyde (PFA)-PBS at 4 C, then cryoprotected overnight in 15% sucrose-PBS and 30% sucrose-PBS, successively, at 4 C.
Trimmed tissues were then flash frozen by dry ice-cooled isopentane in optimal cutting temperature (OCT.) medium (Sakura Finetek). Cryosections (181.tm thickness) were prepared with a Leica CM1860 UV cryostat (Leica Biosystems) and mounted on slides (Superfrost Plus;
Thermo Fisher Scientific, Waltham, MA) with VECTASHIELD with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA), and CytoSeal-60 coverslip sealant (Thermo Fisher Scientific). Confocal imaging was performed using a Zeiss L5M780 laser confocal microscope (Carl Zeiss Jena, Jena, Germany).
For tissue sections stained with lotus tetragonolobus lectin (LTL), the slides containing tissue sections were washed with PBS, treated with 5% normal goat serum (Abcam Catalog # ab7481) and 0.5% IGEPAL CA-630 (Sigma 18896) dissolved in PBS
blocking buffer for 1 hour at room temperature. The slides were then incubated with a 1:100 dilution of biotinylated LTL (Vector Laboratories Cat. No: B-1325) overnight at 4 C. The .. slides were washed and then incubated with a 1:200 dilution of streptavidin-Alexa Fluor 647 (Invitrogen Catalog # S21374) at room temperature for one hour. The slides were washed and coverslips were mounted using Vectashield (without DAPI).
Transgenic mice LSL-Luc mice (Stock No: 005125) and mT/mG mice (Stock No: 007576) were originally purchased from The Jackson Laboratory. Pkdr'c mice of 129S6 genetic background, which develop polycystic kidney disease, were backcrossed with mT/mG mice until pups were acquired that had exactly two copies of the Pkdrc allele and at least one copy of the mT/mG allele, which was confirmed via PCR genotyping.

Example 5: AAV Serotypes and Transduction of Renal Tubule Epithelial Cells after Intravenous Administration Results To examine the ability of AAV vectors to deliver genes into different tissues and the kidney, different AAV serotypes were used to package the Cre recombinase gene.
These vectors were then used to infect cre-reporter luciferase and membrane-bound GFP (mGFP) mice by intravenous injection (Figure 30). Luciferase imaging of living animals demonstrated the ability of different AAV-Cre serotypes to activate luciferase in the liver and other tissues (Figure 31A).
Tissues were collected from these animals and tissue- and cell-specific gene delivery was assessed by observing the conversion of membrane-targeted red fluorescent protein (mRFP)-positive cells that were converted to mGFP-positive cells by Cre by confocal microscopy of tissue sections (Figures 31B to 33). These data indicate that all AAVs have some level of transduction in multiple tissues, but with biases (Figure 31B).
When kidney sections were examined, the pattern of gene delivery as evidenced by mGFP
localization was different by different serotypes (Figure 32A). Globular patterns of mRFP-positive cells in the sections identify the glomerulus within these kidney sections (Figure 32A-E).
Observation of GFP-positive cells within these mRFP glomeruli demonstrates successful delivery of Cre recombinase to either endothelial cells or to podocytes within the glomerulus.
GFP-positive cells outside of the mRFP-positive glomeruli indicate delivery to other renal cells.
When tissue sections were counterstained with cell-specific markers, AAV1 delivery localized with alpha-actin-positive smooth muscle cells in blood vessels rather than in glomerular cells. AAV1 also did not activate mGFP in Lotus Toxin Agglutin (LTA)-positive renal tubules cells (Figure 32B).
AAV8 mediated Cre delivery to glomerular cells as well as macula densa cells, but not to alpha-actin positive smooth muscle cells and not to LTA-positive tubule cells (Figure 32C).

AAV9 mediated Cre delivery to glomerular and macula densa cells, but not to alpha-actin positive smooth muscle cells, nor to alpha-synaptopodin (aSynapt)-positive podocytes, nor to LTA-positive tubule cells, but there was some delivery to EpCAM-positive proximal tubule cells (Figure 32D).
AAVrh10 mediated Cre delivery to glomerular and macula densa cells including CD31-positive glomerular endothelial cells, but not to alpha-actin positive smooth muscle cells, nor to LTA-positive tubule cells (Figure 32E). When CD31-stained glomeruli were examined at higher resolution, it was apparent that AAVrh10 was mediating equal transduction to CD31-positive endothelial cells and to CD31-negative podocytes within the glomerulus.
Together these results demonstrate that multiple serotypes of AAV can be used to deliver nucleic acid to cells within the kidneys. These results also demonstrate that different serotypes and different AAV capsids mediate delivery into different subsets of kidney cells.
Methods pAAV-Cre vectors were packaged an adenovirus helper plasmid with the indicated AAV Rep2/Capl, 8, 9, or rh10 plasmids by triple transfection and AAV particles were purified. These were injected intravenously into Cre reporter mice by tail vein injection.
Mice were anesthetized, injected with luciferin, and imaged for luciferase activity. Animals were sacrificed and frozen tissue sections were examined by confocal microscopy with and without counterstaining for cell-specific proteins using fluorescent antibodies.
SEQUENCES
SEQ ID NO:1 PKD1 cDNA
ATGCCGCCCGCCGCGCCCGCCCGCCTGGCGCTGGCCCTGGGCCTGGGCCTGTGGCTCGGGGCGCTGGCGGGGGG
CCCCGGGGGCGCGCCGGGGGGCCCCGGGCGCGGCTGCGGGCCCTGCGAGCCCCCCTGCCTCTGCGGCCCAGCGC
CCGGCGCCGCCTGCCGCGTCAACTGCTCGGGCCGCGGGCTGCGGACGCTCGGTCCCGCGCTGCGCATCCCCGCG
GACGCCACAGCGCTAGACGTCTCCCACAACCTGCTCCGGGCGCTGGACGTTGGGCTCCTGGCGAACCTCTCGGC
GCTGGCAGAGCTGGATATAAGCAACAACAAGATTTCTACGTTAGAAGAAGGAATATTTGCTAATTTATTTAATT
TAAGTGAAATAAACCTGAGTGGGAACCCGTTTGAGTGTGACTGTGGCCTGGCGTGGCTGCCGCGATGGGCGGAG

GAGCAGCAGGTGCGGGTGGTGCAGCCCGAGGCAGCCACGTGTGCTGGGCCTGGCTCCCTGGCTGGCCAGCCTCT
GCTTGGCATCCCCTTGCTGGACAGTGGCTGTGGTGAGGAGTATGTCGCCTGCCTCCCTGACAACAGCTCAGGCA
CCGTGGCAGCAGTGTCCTTTTCAGCTGCCCACGAAGGCCTGCTTCAGCCAGAGGCCTGCAGCGCCTTCTGCTTC
TCCACCGGCCAGGGCCTCGCAGCCCTCTCGGAGCAGGGCTGGTGCCTGTGTGGGGCGGCCCAGCCCTCCAGTGC
CTCCTTTGCCTGCCTGTCCCTCTGCTCCGGCCCCCCGCCACCTCCTGCCCCCACCTGTAGGGGCCCCACCCTCC
TCCAGCACGTCTTCCCTGCCTCCCCAGGGGCCACCCTGGTGGGGCCCCACGGACCTCTGGCCTCTGGCCAGCTA
GCAGCCTTCCACATCGCTGCCCCGCTCCCTGTCACTGCCACACGCTGGGACTTCGGAGACGGCTCCGCCGAGGT
GGATGCCGCTGGGCCGGCTGCCTCGCATCGCTATGTGCTGCCTGGGCGCTATCACGTGACGGCCGTGCTGGCCC
TGGGGGCCGGCTCAGCCCTGCTGGGGACAGACGTGCAGGTGGAAGCGGCACCTGCCGCCCTGGAGCTCGTGTGC
CCGTCCTCGGTGCAGAGTGACGAGAGCCTTGACCTCAGCATCCAGAACCGCGGTGGTTCAGGCCTGGAGGCCGC
CTACAGCATCGTGGCCCTGGGCGAGGAGCCGGCCCGAGCGGTGCACCCGCTCTGCCCCTCGGACACGGAGATCT
TCCCTGGCAACGGGCACTGCTACCGCCTGGTGGTGGAGAAGGCGGCCTGGCTGCAGGCGCAGGAGCAGTGTCAG
GCCTGGGCCGGGGCCGCCCTGGCAATGGTGGACAGTCCCGCCGTGCAGCGCTTCCTGGTCTCCCGGGTCACCAG
GAGCCTAGACGTGTGGATCGGCTTCTCGACTGTGCAGGGGGTGGAGGTGGGCCCAGCGCCGCAGGGCGAGGCCT
TCAGCCTGGAGAGCTGCCAGAACTGGCTGCCCGGGGAGCCACACCCAGCCACAGCCGAGCACTGCGTCCGGCTC
GGGCCCACCGGGTGGTGTAACACCGACCTGTGCTCAGCGCCGCACAGCTACGTCTGCGAGCTGCAGCCCGGAGG
CCCAGTGCAGGATGCCGAGAACCTCCTCGTGGGAGCGCCCAGTGGGGACCTGCAGGGACCCCTGACGCCTCTGG
CACAGCAGGACGGCCTCTCAGCCCCGCACGAGCCCGTGGAGGTCATGGTATTCCCGGGCCTGCGTCTGAGCCGT
GAAGCCTTCCTCACCACGGCCGAATTTGGGACCCAGGAGCTCCGGCGGCCCGCCCAGCTGCGGCTGCAGGTGTA
CCGGCTCCTCAGCACAGCAGGGACCCCGGAGAACGGCAGCGAGCCTGAGAGCAGGTCCCCGGACAACAGGACCC
AGCTGGCCCCCGCGTGCATGCCAGGGGGACGCTGGTGCCCTGGAGCCAACATCTGCTTGCCGCTGGACGCCTCC
TGCCACCCCCAGGCCTGCGCCAATGGCTGCACGTCAGGGCCAGGGCTACCCGGGGCCCCCTATGCGCTATGGAG
AGAGTTCCTCTTCTCCGTTCCCGCGGGGCCCCCCGCGCAGTACTCGGTCACCCTCCACGGCCAGGATGTCCTCA
TGCTCCCTGGTGACCTCGTTGGCTTGCAGCACGACGCTGGCCCTGGCGCCCTCCTGCACTGCTCGCCGGCTCCC
GGCCACCCTGGTCCCCAGGCCCCGTACCTCTCCGCCAACGCCTCGTCATGGCTGCCCCACTTGCCAGCCCAGCT
GGAGGGCACTTGGGCCTGCCCTGCCTGTGCCCTGCGGCTGCTTGCAGCCACGGAACAGCTCACCGTGCTGCTGG
GCTTGAGGCCCAACCCTGGACTGCGGCTGCCTGGGCGCTATGAGGTCCGGGCAGAGGTGGGCAATGGCGTGTCC
AGGCACAACCTCTCCTGCAGCTTTGACGTGGTCTCCCCAGTGGCTGGGCTGCGGGTCATCTACCCTGCCCCCCG
CGACGGCCGCCTCTACGTGCCCACCAACGGCTCAGCCTTGGTGCTCCAGGTGGACTCTGGTGCCAACGCCACGG
CCACGGCTCGCTGGCCTGGGGGCAGTGTCAGCGCCCGCTTTGAGAATGTCTGCCCTGCCCTGGTGGCCACCTTC
GTGCCCGGCTGCCCCTGGGAGACCAACGATACCCTGTTCTCAGTGGTAGCACTGCCGTGGCTCAGTGAGGGGGA
GCACGTGGTGGACGTGGTGGTGGAAAACAGCGCCAGCCGGGCCAACCTCAGCCTGCGGGTGACGGCGGAGGAGC
CCATCTGTGGCCTCCGCGCCACGCCCAGCCCCGAGGCCCGTGTACTGCAGGGAGTCCTAGTGAGGTACAGCCCC
GTGGTGGAGGCCGGCTCGGACATGGTCTTCCGGTGGACCATCAACGACAAGCAGTCCCTGACCTTCCAGAACGT
GGTCTTCAATGTCATTTATCAGAGCGCGGCGGTCTTCAAGCTCTCACTGACGGCCTCCAACCACGTGAGCAACG
TCACCGTGAACTACAACGTAACCGTGGAGCGGATGAACAGGATGCAGGGTCTGCAGGTCTCCACAGTGCCGGCC

GTGCTGTCCCCCAATGCCACGCTAGCACTGACGGCGGGCGTGCTGGTGGACTCGGCCGTGGAGGTGGCCTTCCT
GTGGACCTTTGGGGATGGGGAGCAGGCCCTCCACCAGTTCCAGCCTCCGTACAACGAGTCCTTCCCGGTTCCAG
ACCCCTCGGTGGCCCAGGTGCTGGTGGAGCACAATGTCATGCACACCTACGCTGCCCCAGGTGAGTACCTCCTG
ACCGTGCTGGCATCTAATGCCTTCGAGAACCTGACGCAGCAGGTGCCTGTGAGCGTGCGCGCCTCCCTGCCCTC
CGTGGCTGTGGGTGTGAGTGACGGCGTCCTGGTGGCCGGCCGGCCCGTCACCTTCTACCCGCACCCGCTGCCCT
CGCCTGGGGGTGTTCTTTACACGTGGGACTTCGGGGACGGCTCCCCTGTCCTGACCCAGAGCCAGCCGGCTGCC
AACCACACCTATGCCTCGAGGGGCACCTACCACGTGCGCCTGGAGGTCAACAACACGGTGAGCGGTGCGGCGGC
CCAGGCGGATGTGCGCGTCTTTGAGGAGCTCCGCGGACTCAGCGTGGACATGAGCCTGGCCGTGGAGCAGGGCG
CCCCCGTGGTGGTCAGCGCCGCGGTGCAGACGGGCGACAACATCACGTGGACCTTCGACATGGGGGACGGCACC
GTGCTGTCGGGCCCGGAGGCAACAGTGGAGCATGTGTACCTGCGGGCACAGAACTGCACAGTGACCGTGGGTGC
GGCCAGCCCCGCCGGCCACCTGGCCCGGAGCCTGCACGTGCTGGTCTTCGTCCTGGAGGTGCTGCGCGTTGAAC
CCGCCGCCTGCATCCCCACGCAGCCTGACGCGCGGCTCACGGCCTACGTCACCGGGAACCCGGCCCACTACCTC
TTCGACTGGACCTTCGGGGATGGCTCCTCCAACACGACCGTGCGGGGGTGCCCGACGGTGACACACAACTTCAC
GCGGAGCGGCACGTTCCCCCTGGCGCTGGTGCTGTCCAGCCGCGTGAACAGGGCGCATTACTTCACCAGCATCT
GCGTGGAGCCAGAGGTGGGCAACGTCACCCTGCAGCCAGAGAGGCAGTTTGTGCAGCTCGGGGACGAGGCCTGG
CTGGTGGCATGTGCCTGGCCCCCGTTCCCCTACCGCTACACCTGGGACTTTGGCACCGAGGAAGCCGCCCCCAC
CCGTGCCAGGGGCCCTGAGGTGACGTTCATCTACCGAGACCCAGGCTCCTATCTTGTGACAGTCACCGCGTCCA
ACAACATCTCTGCTGCCAATGACTCAGCCCTGGTGGAGGTGCAGGAGCCCGTGCTGGTCACCAGCATCAAGGTC
AATGGCTCCCTTGGGCTGGAGCTGCAGCAGCCGTACCTGTTCTCTGCTGTGGGCCGTGGGCGCCCCGCCAGCTA
CCTGTGGGATCTGGGGGACGGTGGGTGGCTCGAGGGTCCGGAGGTCACCCACGCTTACAACAGCACAGGTGACT
TCACCGTTAGGGTGGCCGGCTGGAATGAGGTGAGCCGCAGCGAGGCCTGGCTCAATGTGACGGTGAAGCGGCGC
GTGCGGGGGCTCGTCGTCAATGCAAGCCGCACGGTGGTGCCCCTGAATGGGAGCGTGAGCTTCAGCACGTCGCT
GGAGGCCGGCAGTGATGTGCGCTATTCCTGGGTGCTCTGTGACCGCTGCACGCCCATCCCTGGGGGTCCTACCA
TCTCTTACACCTTCCGCTCCGTGGGCACCTTCAATATCATCGTCACGGCTGAGAACGAGGTGGGCTCCGCCCAG
GACAGCATCTTCGTCTATGTCCTGCAGCTCATAGAGGGGCTGCAGGTGGTGGGCGGTGGCCGCTACTTCCCCAC
CAACCACACGGTACAGCTGCAGGCCGTGGTTAGGGATGGCACCAACGTCTCCTACAGCTGGACTGCCTGGAGGG
ACAGGGGCCCGGCCCTGGCCGGCAGCGGCAAAGGCTTCTCGCTCACCGTGCTCGAGGCCGGCACCTACCATGTG
CAGCTGCGGGCCACCAACATGCTGGGCAGCGCCTGGGCCGACTGCACCATGGACTTCGTGGAGCCTGTGGGGTG
GCTGATGGTGGCCGCCTCCCCGAACCCAGCTGCCGTCAACACAAGCGTCACCCTCAGTGCCGAGCTGGCTGGTG
GCAGTGGTGTCGTATACACTTGGTCCTTGGAGGAGGGGCTGAGCTGGGAGACCTCCGAGCCATTTACCACCCAT
AGCTTCCCCACACCCGGCCTGCACTTGGTCACCATGACGGCAGGGAACCCGCTGGGCTCAGCCAACGCCACCGT
GGAAGTGGATGTGCAGGTGCCTGTGAGTGGCCTCAGCATCAGGGCCAGCGAGCCCGGAGGCAGCTTCGTGGCGG
CCGGGTCCTCTGTGCCCTTTTGGGGGCAGCTGGCCACGGGCACCAATGTGAGCTGGTGCTGGGCTGTGCCCGGC
GGCAGCAGCAAGCGTGGCCCTCATGTCACCATGGTCTTCCCGGATGCTGGCACCTTCTCCATCCGGCTCAATGC
CTCCAACGCAGTCAGCTGGGTCTCAGCCACGTACAACCTCACGGCGGAGGAGCCCATCGTGGGCCTGGTGCTGT
GGGCCAGCAGCAAGGTGGTGGCGCCCGGGCAGCTGGTCCATTTTCAGATCCTGCTGGCTGCCGGCTCAGCTGTC

ACCTTCCGCCTGCAGGTCGGCGGGGCCAACCCCGAGGTGCTCCCCGGGCCCCGTTTCTCCCACAGCTTCCCCCG
CGTCGGAGACCACGTGGTGAGCGTGCGGGGCAAAAACCACGTGAGCTGGGCCCAGGCGCAGGTGCGCATCGTGG
TGCTGGAGGCCGTGAGTGGGCTGCAGGTGCCCAACTGCTGCGAGCCTGGCATCGCCACGGGCACTGAGAGGAAC
TTCACAGCCCGCGTGCAGCGCGGCTCTCGGGTCGCCTACGCCTGGTACTTCTCGCTGCAGAAGGTCCAGGGCGA
CTCGCTGGTCATCCTGTCGGGCCGCGACGTCACCTACACGCCCGTGGCCGCGGGGCTGTTGGAGATCCAGGTGC
GCGCCTTCAACGCCCTGGGCAGTGAGAACCGCACGCTGGTGCTGGAGGTTCAGGACGCCGTCCAGTATGTGGCC
CTGCAGAGCGGCCCCTGCTTCACCAACCGCTCGGCGCAGTTTGAGGCCGCCACCAGCCCCAGCCCCCGGCGTGT
GGCCTACCACTGGGACTTTGGGGATGGGTCGCCAGGGCAGGACACAGATGAGCCCAGGGCCGAGCACTCCTACC
TGAGGCCTGGGGACTACCGCGTGCAGGTGAACGCCTCCAACCTGGTGAGCTTCTTCGTGGCGCAGGCCACGGTG
ACCGTCCAGGTGCTGGCCTGCCGGGAGCCGGAGGTGGACGTGGTCCTGCCCCTGCAGGTGCTGATGCGGCGATC
ACAGCGCAACTACTTGGAGGCCCACGTTGACCTGCGCGACTGCGTCACCTACCAGACTGAGTACCGCTGGGAGG
TGTATCGCACCGCCAGCTGCCAGCGGCCGGGGCGCCCAGCGCGTGTGGCCCTGCCCGGCGTGGACGTGAGCCGG
CCTCGGCTGGTGCTGCCGCGGCTGGCGCTGCCTGTGGGGCACTACTGCTTTGTGTTTGTCGTGTCATTTGGGGA
CACGCCACTGACACAGAGCATCCAGGCCAATGTGACGGTGGCCCCCGAGCGCCTGGTGCCCATCATTGAGGGTG
GCTCATACCGCGTGTGGTCAGACACACGGGACCTGGTGCTGGATGGGAGCGAGTCCTACGACCCCAACCTGGAG
GACGGCGACCAGACGCCGCTCAGTTTCCACTGGGCCTGTGTGGCTTCGACACAGAGGGAGGCTGGCGGGTGTGC
GCTGAACTTTGGGCCCCGCGGGAGCAGCACGGTCACCATTCCACGGGAGCGGCTGGCGGCTGGCGTGGAGTACA
CCTTCAGCCTGACCGTGTGGAAGGCCGGCCGCAAGGAGGAGGCCACCAACCAGACGGTGCTGATCCGGAGTGGC
CGGGTGCCCATTGTGTCCTTGGAGTGTGTGTCCTGCAAGGCACAGGCCGTGTACGAAGTGAGCCGCAGCTCCTA
CGTGTACTTGGAGGGCCGCTGCCTCAATTGCAGCAGCGGCTCCAAGCGAGGGCGGTGGGCTGCACGTACGTTCA
GCAACAAGACGCTGGTGCTGGATGAGACCACCACATCCACGGGCAGTGCAGGCATGCGACTGGTGCTGCGGCGG
GGCGTGCTGCGGGACGGCGAGGGATACACCTTCACGCTCACGGTGCTGGGCCGCTCTGGCGAGGAGGAGGGCTG
CGCCTCCATCCGCCTGTCCCCCAACCGCCCGCCGCTGGGGGGCTCTTGCCGCCTCTTCCCACTGGGCGCTGTGC
ACGCCCTCACCACCAAGGTGCACTTCGAATGCACGGGCTGGCATGACGCGGAGGATGCTGGCGCCCCGCTGGTG
TACGCCCTGCTGCTGCGGCGCTGTCGCCAGGGCCACTGCGAGGAGTTCTGTGTCTACAAGGGCAGCCTCTCCAG
CTACGGAGCCGTGCTGCCCCCGGGTTTCAGGCCACACTTCGAGGTGGGCCTGGCCGTGGTGGTGCAGGACCAGC
TGGGAGCCGCTGTGGTCGCCCTCAACAGGTCTTTGGCCATCACCCTCCCAGAGCCCAACGGCAGCGCAACGGGG
CTCACAGTCTGGCTGCACGGGCTCACCGCTAGTGTGCTCCCAGGGCTGCTGCGGCAGGCCGATCCCCAGCACGT
CATCGAGTACTCGTTGGCCCTGGTCACCGTGCTGAACGAGTACGAGCGGGCCCTGGACGTGGCGGCAGAGCCCA
AGCACGAGCGGCAGCACCGAGCCCAGATACGCAAGAACAT CACGGAGACT CT GGT GT CCCT GAGGGT
CCACACT
GTGGATGACATCCAGCAGATCGCTGCTGCGCTGGCCCAGTGCATGGGGCCCAGCAGGGAGCTCGTATGCCGCTC
GTGCCTGAAGCAGACGCTGCACAAGCTGGAGGCCATGATGCTCATCCTGCAGGCAGAGACCACCGCGGGCACCG
TGACGCCCACCGCCATCGGAGACAGCATCCTCAACATCACAGGAGACCTCATCCACCTGGCCAGCTCGGACGTG
CGGGCACCACAGCCCTCAGAGCTGGGAGCCGAGTCACCATCTCGGATGGTGGCGTCCCAGGCCTACAACCTGAC
CTCTGCCCTCATGCGCATCCTCATGCGCTCCCGCGTGCTCAACGAGGAGCCCCTGACGCTGGCGGGCGAGGAGA
TCGTGGCCCAGGGCAAGCGCTCGGACCCGCGGAGCCTGCTGTGCTATGGCGGCGCCCCAGGGCCTGGCTGCCAC

TTCTCCATCCCCGAGGCTTTCAGCGGGGCCCTGGCCAACCTCAGTGACGTGGTGCAGCTCATCTTTCTGGTGGA
CTCCAATCCCTTTCCCTTTGGCTATATCAGCAACTACACCGTCTCCACCAAGGTGGCCTCGATGGCATTCCAGA
CACAGGCCGGCGCCCAGATCCCCATCGAGCGGCTGGCCTCAGAGCGCGCCATCACCGTGAAGGTGCCCAACAAC
TCGGACTGGGCTGCCCGGGGCCACCGCAGCTCCGCCAACTCCGCCAACTCCGTTGTGGTCCAGCCCCAGGCCTC
CGTCGGTGCTGTGGTCACCCTGGACAGCAGCAACCCTGCGGCCGGGCTGCATCTGCAGCTCAACTATACGCTGC
TGGACGGCCACTACCTGTCTGAGGAACCTGAGCCCTACCTGGCAGTCTACCTACACTCGGAGCCCCGGCCCAAT
GAGCACAACTGCTCGGCTAGCAGGAGGATCCGCCCAGAGTCACTCCAGGGTGCTGACCACCGGCCCTACACCTT
CTTCATTTCCCCGGGGAGCAGAGACCCAGCGGGGAGTTACCATCTGAACCTCTCCAGCCACTTCCGCTGGTCGG
CGCTGCAGGTGTCCGTGGGCCTGTACACGTCCCTGTGCCAGTACTTCAGCGAGGAGGACATGGTGTGGCGGACA
GAGGGGCTGCTGCCCCTGGAGGAGACCTCGCCCCGCCAGGCCGTCTGCCTCACCCGCCACCTCACCGCCTTCGG
CGCCAGCCTCTTCGTGCCCCCAAGCCATGTCCGCTTTGTGTTTCCTGAGCCGACAGCGGATGTAAACTACATCG
TCATGCTGACATGTGCTGTGTGCCTGGTGACCTACATGGTCATGGCCGCCATCCTGCACAAGCTGGACCAGTTG
GATGCCAGCCGGGGCCGCGCCATCCCTTTCTGTGGGCAGCGGGGCCGCTTCAAGTACGAGATCCTCGTCAAGAC
AGGCTGGGGCCGGGGCTCAGGTACCACGGCCCACGTGGGCATCATGCTGTATGGGGTGGACAGCCGGAGCGGCC
ACCGGCACCTGGACGGCGACAGAGCCTTCCACCGCAACAGCCTGGACATCTTCCGGATCGCCACCCCGCACAGC
CTGGGTAGCGTGTGGAAGATCCGAGTGTGGCACGACAACAAAGGGCTCAGCCCTGCCTGGTTCCTGCAGCACGT
CATCGTCAGGGACCTGCAGACGGCACGCAGCGCCTTCTTCCTGGTCAATGACTGGCTTTCGGTGGAGACGGAGG
CCAACGGGGGCCTGGTGGAGAAGGAGGTGCTGGCCGCGAGCGACGCAGCCCTTTTGCGCTTCCGGCGCCTGCTG
GTGGCTGAGCTGCAGCGTGGCTTCTTTGACAAGCACATCTGGCTCTCCATATGGGACCGGCCGCCTCGTAGCCG
TTTCACTCGCATCCAGAGGGCCACCTGCTGCGTTCTCCTCATCTGCCTCTTCCTGGGCGCCAACGCCGTGTGGT
ACGGGGCTGTTGGCGACTCTGCCTACAGCACGGGGCATGTGTCCAGGCTGAGCCCGCTGAGCGTCGACACAGTC
GCTGTTGGCCTGGTGTCCAGCGTGGTTGTCTATCCCGTCTACCTGGCCATCCTTTTTCTCTTCCGGATGTCCCG
GAGCAAGGTGGCTGGGAGCCCGAGCCCCACACCTGCCGGGCAGCAGGTGCTGGACATCGACAGCTGCCTGGACT
CGTCCGTGCTGGACAGCTCCTTCCTCACGTTCTCAGGCCTCCACGCTGAGCAGGCCTTTGTTGGACAGATGAAG
AGTGACTTGTTTCTGGATGATTCTAAGAGTCTGGTGTGCTGGCCCTCCGGCGAGGGAACGCTCAGTTGGCCGGA
CCTGCTCAGTGACCCGTCCATTGTGGGTAGCAATCTGCGGCAGCTGGCACGGGGCCAGGCGGGCCATGGGCTGG
GCCCAGAGGAGGACGGCTTCTCCCTGGCCAGCCCCTACTCGCCTGCCAAATCCTTCTCAGCATCAGATGAAGAC
CTGATCCAGCAGGTCCTTGCCGAGGGGGTCAGCAGCCCAGCCCCTACCCAAGACACCCACATGGAAACGGACCT
GCTCAGCAGCCTGTCCAGCACTCCTGGGGAGAAGACAGAGACGCTGGCGCTGCAGAGGCTGGGGGAGCTGGGGC
CACCCAGCCCAGGCCTGAACTGGGAACAGCCCCAGGCAGCGAGGCTGTCCAGGACAGGACTGGTGGAGGGTCTG
CGGAAGCGCCTGCTGCCGGCCTGGTGTGCCTCCCTGGCCCACGGGCTCAGCCTGCTCCTGGTGGCTGTGGCTGT
GGCTGTCTCAGGGTGGGTGGGTGCGAGCTTCCCCCCGGGCGTGAGTGTTGCGTGGCTCCTGTCCAGCAGCGCCA
GCTTCCTGGCCTCATTCCTCGGCTGGGAGCCACTGAAGGTCTTGCTGGAAGCCCTGTACTTCTCACTGGTGGCC
AAGCGGCTGCACCCGGATGAAGATGACACCCTGGTAGAGAGCCCGGCTGTGACGCCTGTGAGCGCACGTGTGCC
CCGCGTACGGCCACCCCACGGCTTTGCACTCTTCCTGGCCAAGGAAGAAGCCCGCAAGGTCAAGAGGCTACATG
GCATGCTGCGGAGCCTCCTGGTGTACATGCTTTTTCTGCTGGTGACCCTGCTGGCCAGCTATGGGGATGCCTCA

T GCCAT GGGCACGCCTACCGT CT GCAAAGCGCCAT CAAGCAGGAGCT GCACAGCCGGGCCTT CCT
GGCCAT CAC
GCGGT CT GAGGAGCT CT GGCCAT GGAT GGCCCACGT GCT GCT GCCCTACGT CCACGGGAACCAGT
CCAGCCCAG
AGCTGGGGCCCCCACGGCTGCGGCAGGTGCGGCTGCAGGAAGCACTCTACCCAGACCCTCCCGGCCCCAGGGTC
CACACGTGCTCGGCCGCAGGAGGCTTCAGCACCAGCGATTACGACGTTGGCTGGGAGAGTCCTCACAATGGCTC
GGGGACGT GGGCCTATT CAGCGCCGGAT CT GCT GGGGGCAT GGT CCT GGGGCT CCT GT GCCGT
GTAT GACAGCG
GGGGCTACGTGCAGGAGCTGGGCCTGAGCCTGGAGGAGAGCCGCGACCGGCTGCGCTTCCTGCAGCTGCACAAC
T GGCT GGACAACAGGAGCCGCGCT GT GTT CCT GGAGCT CACGCGCTACAGCCCGGCCGT GGGGCT
GCACGCCGC
CGT CACGCT GCGCCT CGAGTT CCCGGCGGCCGGCCGCGCCCT GGCCGCCCT CAGCGT CCGCCCCTTT
GCGCT GC
GCCGCCT CAGCGCGGGCCT CT CGCT GCCT CT GCT CACCT CGGT GT GCCT GCT GCT GTT CGCCGT
GCACTT CGCC
GT GGCCGAGGCCCGTACTT GGCACAGGGAAGGGCGCT GGCGCGT GCT GCGGCT CGGAGCCT GGGCGCGGT
GGCT
GCTGGTGGCGCTGACGGCGGCCACGGCACTGGTACGCCTCGCCCAGCTGGGTGCCGCTGACCGCCAGTGGACCC
GTTTCGTGCGCGGCCGCCCGCGCCGCTTCACTAGCTTCGACCAGGTGGCGCAGCTGAGCTCCGCAGCCCGTGGC
CT GGCGGCCT CGCT GCT CTT CCT GCTTTT GGT CAAGGCT GCCCAGCAGCTACGCTT CGT GCGCCAGT
GGT CCGT
CTTT GGCAAGACATTAT GCCGAGCT CT GCCAGAGCT CCT GGGGGT CACCTT GGGCCT GGT GGT GCT
CGGGGTAG
CCTACGCCCAGCT GGCCAT CCT GCT CGT GT CTT CCT GT GT GGACT CCCT CT GGAGCGT
GGCCCAGGCCCT GTT G
GT GCT GT GCCCT GGGACT GGGCT CT CTACCCT GT GT CCT GCCGAGT CCT GGCACCT GT
CACCCCT GCT GT GT GT
GGGGCT CT GGGCACT GCGGCT GT GGGGCGCCCTACGGCT GGGGGCT GTTATT CT CCGCT
GGCGCTACCACGCCT
TGCGTGGAGAGCTGTACCGGCCGGCCTGGGAGCCCCAGGACTACGAGATGGTGGAGTTGTTCCTGCGCAGGCTG
CGCCT CT GGAT GGGCCT CAGCAAGGT CAAGGAGTT CCGCCACAAAGT CCGCTTT GAAGGGAT
GGAGCCGCT GCC
CT CT CGCT CCT CCAGGGGCT CCAAGGTAT CCCCGGAT GT GCCCCCACCCAGCGCT GGCT CCGAT
GCCT CGCACC
CCT CCACCT CCT CCAGCCAGCT GGAT GGGCT GAGCGT GAGCCT GGGCCGGCT GGGGACAAGGT GT
GAGCCT GAG
CCCTCCCGCCTCCAAGCCGTGTTCGAGGCCCTGCTCACCCAGTTTGACCGACTCAACCAGGCCACAGAGGACGT
CTACCAGCTGGAGCAGCAGCTGCACAGCCTGCAAGGCCGCAGGAGCAGCCGGGCGCCCGCCGGATCTTCCCGTG
GCCCAT CCCCGGGCCT GCGGCCAGCACT GCCCAGCCGCCTT GCCCGGGCCAGT CGGGGT GT GGACCT
GGCCACT
GGCCCCAGCAGGACACCCCTTCGGGCCAAGAACAAGGTCCACCCCAGCAGCACTTAG
SEQ ID NO:2 PC-1 polypeptide MP PAAPARLALALGLGLWLGALAGGP GGAP GGP GRGCGP CEP P CLCGPAP GAACRVNCS GRGLRT
LGPALRI PA
DATALDVSHNLLRALDVGLLANLSALAELDI SNNKI STLEEGI FANL FNL S EINL S GNP FECDCGLAWL
P RWAE
EQQVRVVQP EAAT CAGP GS LAGQP LLGI P LLDS GCGEEYVACL P DNS SGTVAAVS
FSAAHEGLLQPEACSAFCF
STGQGLAALSEQGWCLCGAAQPS SAS FACL S LCS GP P P P PAPT CRGPT LLQHVFPAS P GAT
LVGPHGP LAS GQL
AAFHIAAP L PVTAT RWDFGDGSAEVDAAGPAASHRYVL P GRYHVTAVLAL GAGSALLGT
DVQVEAAPAALELVC
PS SVQ S DES LDL S I QNRGGS GLEAAYS IVALGEEPARAVHP LCP S DT EI FP
GNGHCYRLVVEKAAWLQAQEQCQ
AWAGAALAMVDS PAVQRFLVS RVT RS LDVWI GFS TVQGVEVGPAPQGEAFS LE S CQNWL P GE PH
PATAEHCVRL
GPT GWCNT DLCSAPHS YVCELQP GGPVQDAENLLVGAP S GDLQGP LT P LAQQDGL SAPHEPVEVMVFP
GLRL S R

EAFLTTAEFGTQELRRPAQLRLQVYRLLSTAGT P ENGS EP ES RS P DNRTQLAPACMP GGRWC P GANT
CLPLDAS
CH PQACANGCT S GP GL P GAP YALWRE FL FSVPAGP
PAQYSVTLHGQDVLMLPGDLVGLQHDAGPGALLHCS PAP
GH P GPQAP YL SANAS
SWLPHLPAQLEGTWACPACALRLLAATEQLTVLLGLRPNPGLRLPGRYEVRAEVGNGVS
RHNLS CS FDVVS PVAGLRVI YPAPRDGRLYVPTNGSALVLQVDS GANATATARWPGGSVSARFENVCPALVAT
F
VP GC PWETNDT L FSVVAL PWL S EGEHVVDVVVEN SAS RANL S LRVTAEE P I CGLRAT P S
PEARVLQGVLVRYS P
VVEAGSDMVFRWT I NDKQ S LT FQNVVENVI
YQSAAVFKLSLTASNHVSNVTVNYNVTVERMNRMQGLQVSTVPA
VLS PNATLALTAGVLVDSAVEVAFLWT FGDGEQALHQFQP PYNES FPVPDP
SVAQVLVEHNVMHTYAAPGEYLL
TVLASNAFENLTQQVPVSVRASLP SVAVGVSDGVLVAGRPVT FYPHPLPSPGGVLYTWDFGDGS PVLTQSQPAA
NHTYASRGTYHVRLEVNNTVS GAAAQADVRVFEELRGL SVDMS LAVEQGAPVVVSAAVQT GDN I TWT
FDMGDGT
VLS GP EATVEHVYL RAQN CTVTVGAAS PAGH LARS LHVLVFVL EVL RVE PAAC I
PTQPDARLTAYVTGNPAHYL
FDWT FGDGS SNT TVRGC P TVTHNFT RS GT FP LALVL S SRVNRAHYFT S I
CVEPEVGNVTLQPERQFVQLGDEAW
LVACAWP P FP YRYTWD FGT EEAAP T RARGP EVT FI YRD P GS YLVTVTASNN I
SAANDSALVEVQEPVLVT S I KV
NGS L GLELQQ P YL FSAVGRGRPAS YLWDL GDGGWLEGP EVTHAYN S T GD FTVRVAGWNEVS RS
EAWLNVTVKRR
VRGLVVNASRTVVPLNGSVS FS T SLEAGSDVRYSWVLCDRCT P I PGGPT I SYT FRSVGT FNI
IVTAENEVGSAQ
DS I FVYVLQL I EGLQVVGGGRYFPTNHTVQLQAVVRDGTNVSYSWTAWRDRGPALAGS
GKGFSLTVLEAGTYHV
QLRATNMLGSAWADCTMDFVEPVGWLMVAAS PNPAAVNT S VT L SAELAGGS GVVYTWSLEEGLSWETS EP
FT TH
S FP T PGLHLVTMTAGNPLGSANATVEVDVQVPVS GLS I RAS E P GGS FVAAGS SVP
FWGQLATGTNVSWCWAVPG
GS SKRGPHVTMVFPDAGT FS I RLNASNAVSWVSATYNLTAEEP IVGLVLWAS S KVVAP GQLVH FQ I
LLAAGSAV
T FRLQVGGANP EVL P GP RFS H S FP RVGDHVVSVRGKNHVSWAQAQVRIVVLEAVS GLQVPNCCE P
GIAT GT ERN
FTARVQRGSRVAYAWYFSLQKVQGDSLVI LS GRDVTYT PVAAGLLE I QVRAFNAL GS ENRT
LVLEVQDAVQYVA
LQS GP C FTNRSAQ FEAAT SPSPRRVAYHWDFGDGS P GQDT DE P RAEH S YLRP
GDYRVQVNASNLVS FFVAQATV
TVQVLACREPEVDVVLPLQVLMRRSQRNYLEAHVDLRDCVTYQTEYRWEVYRTAS CQRPGRPARVALPGVDVSR
PRLVLPRLALPVGHYCFVFVVS FGDT PLTQS I QANVTVAP ERLVP I I EGGS YRVWS DT RDLVLDGS
E S YD PNLE
DGDQT PLS FHWACVASTQREAGGCALNFGPRGS STVT I PRERLAAGVEYT FS LTVWKAGRKEEATNQTVL
I RS G
RVP IVSLECVS CKAQAVYEVS RS SYVYLEGRCLNCS S GS KRGRWAART
FSNKTLVLDETTTSTGSAGMRLVLRR
GVLRDGEGYT FT LTVL GRS GEEEGCAS I RLS PNRP PLGGS CRL FP L GAVHALT T KVH FECT
GWHDAEDAGAP LV
YALLLRRCRQGHCEEFCVYKGSLS SYGAVLP P GFRPH FEVGLAVVVQDQL GAAVVALNRS LAI
TLPEPNGSATG
LTVWLHGLTASVLPGLLRQADPQHVI EYS LALVTVLNEYERALDVAAE P KHERQHRAQ I RKNI T ET LVS
LRVHT
VDD I QQ IAAALAQCMGP SRELVCRS CLKQT LHKLEAMML I LQAETTAGTVT PTAI GDS I LNI T
GDL I HLAS S DV
.. RAPQP S EL GAE S P SRMVASQAYNLT SALMRI LMRS RVLNEE P LT LAGEE IVAQGKRS D P
RS LLCYGGAP GP GCH
FS I PEAFS GALANL S DVVQL I FLVD SNP FP FGYI SNYTVS T KVASMAFQTQAGAQ I P I
ERLASERAI TVKVPNN
SDWAARGHRS SAN SAN SVVVQ PQASVGAVVT LD S
SNPAAGLHLQLNYTLLDGHYLSEEPEPYLAVYLHSEPRPN
EHNC SAS RRI RP E S LQGADHRP YT FFI S P GS RD PAGS YHLNL S S H FRWSALQVSVGLYT
SLCQYFSEEDMVWRT
EGLLPLEET S P RQAVCLT RHLTAFGAS L FVP P SHVRFVFPEPTADVNYIVMLTCAVCLVTYMVMAAI
LHKLDQL
.. DAS RGRAI P FCGQRGRFKYE I LVKTGWGRGS GT TAHVGIMLYGVD S RS GHRHLDGDRAFHRN S
LD I FRIAT PH S
LGSVWKI RVWHDNKGLS PAWFLQHVIVRDLQTARSAFFLVNDWL SVET EANGGLVEKEVLAAS
DAALLRFRRLL

VAELQRGFEDKHIWLS IWDRP P RS RFT RI QRAT CCVLL I CL FL GANAVWYGAVGD SAYS T GHVS
RL S PLSVDTV
AVGLVS SVVVYPVYLAI L FL FRMS RS KVAGS P S PT PAGQQVLD I DS CLDS SVLDS S FLT FS
GLHAEQAFVGQMK
S DL FLDD S KS LVCWP S GEGT L SWP DLL S DP S IVGSNLRQLARGQAGHGL GP EEDGFS LAS
PYS PAKS FSASDED
L I QQVLAEGVS S PAP TQDTHMET DLL S S L S S T P GEKT ET LALQRL GEL GP P S
PGLNWEQPQAARLSRTGLVEGL
RKRLLPAWCASLAHGLSLLLVAVAVAVSGWVGAS FP PGVSVAWLLS S SAS FLAS FL GWE P
LKVLLEALYFS LVA
KRLHPDEDDTLVES PAVT PVSARVPRVRP PHGFAL FLAKEEARKVKRLHGMLRS LLVYML FLLVT LLAS
YGDAS
CHGHAYRLQSAI KQELHSRAFLAI T RS EELWPWMAHVLL P YVHGNQ S S P EL GP
PRLRQVRLQEALYPDP P GP RV
HT C SAAGGFS T SDYDVGWES PHNGS GTWAYSAP DLL GAWSWGS CAVYD S GGYVQEL GL S LEE S
RDRLRFLQLHN
WLDNRSRAVFLELTRYS PAVGLHAAVTLRLEFPAAGRALAALSVRP FALRRLSAGLSLPLLT SVCLLL FAVH
FA
VAEARTWH RE GRWRVL RL GAWARWL LVALTAATALVRLAQ L GAAD RQWT RFVRGRP RRFT S
FDQVAQLS SAARG
LAAS LL FLLLVKAAQQLREVRQWSVEGKT LCRAL P ELL GVT L GLVVL GVAYAQLAI LLVS S
CVDSLWSVAQALL
VLC P GT GL S T LC PAE SWHL S PLLCVGLWALRLWGALRLGAVI
LRWRYHALRGELYRPAWEPQDYEMVELFLRRL
RLWMGL S KVKE FRHKVRFEGME P L P S RS SRGSKVS PDVP P P SAGS DAS HP S T S S S
QLDGL SVS L GRL GT RCE P E
P SRLQAVFEALLTQFDRLNQATEDVYQLEQQLHSLQGRRS SRAPAGS SRGP S PGLRPALP
SRLARASRGVDLAT
GP SRT PLRAKNKVHP S ST
SEQ ID NO: .3 PKD2 cDNA
AT GGT GAACT CCAGT CGCGT GCAGCCT
CAGCAGCCCGGGGACGCCAAGCGGCCGCCCGCGCCCCGCGCGCCGGA
CCCGGGCCGGCT GAT GGCT GGCT GCGCGGCCGT GGGCGCCAGCCT CGCCGCCCCGGGCGGCCT CT
GCGAGCAGC
GGGGCCT GGAGAT CGAGAT GCAGCGCAT CCGGCAGGCGGCCGCGCGGGACCCCCCGGCCGGAGCCGCGGCCT
CC
CCT T CT CCT CCGCT CT CGT CGT GCT CCCGGCAGGCGT GGAGCCGCGATAACCCCGGCT T
CGAGGCCGAGGAGGA
GGAGGAGGAGGTGGAAGGGGAAGAAGGCGGAATGGTGGTGGAGATGGACGTAGAGTGGCGCCCGGGCAGCCGGA
GGTCGGCCGCCTCCTCGGCCGTGAGCTCCGTGGGCGCGCGGAGCCGGGGGCTTGGGGGCTACCACGGCGCGGGC
CACCCGAGCGGGAGGCGGCGCCGGCGAGAGGACCAGGGCCCGCCGTGCCCCAGCCCAGTCGGCGGCGGGGACCC
GCT GCAT CGCCACCT CCCCCT GGAAGGGCAGCCGCCCCGAGT GGCCT GGGCGGAGAGGCT GGT T
CGCGGGCT GC
GAGGT CT CT GGGGAACAAGACT CAT GGAGGAAAGCAGCAC TAACCGAGAGAAATACCT TAAAAGT GT T
T TACGG
GAACT GGT CACATACCT CCT T T T T CT CATAGT CT T GT GCAT CT T GACCTACGGCAT GAT
GAGCT CCAAT GT GTA
C TAC TACACCCGGAT GAT GT CACAGCT CT T CCTAGACACCCCCGT GT CCAAAACGGAGAAAAC
TAACT T TAAAA
CT CT GT CT T CCAT GGAAGACT T CT GGAAGT T CACAGAAGGCT CCT TAT T GGAT GGGCT
GTACT GGAAGAT GCAG
CCCAGCAACCAGACT GAAGCT GACAACCGAAGT T T CAT CT T CTAT GAGAACCT GCT GT TAGGGGT
T CCACGAAT
ACGGCAACTCCGAGTCAGAAATGGATCCTGCTCTATCCCCCAGGACTTGAGAGATGAAATTAAAGAGTGCTATG
AT GT CTACT CT GT CAGTAGT GAAGATAGGGCT CCCT T T GGGCCCCGAAAT GGAACCGCT T GGAT
CTACACAAGT
GAAAAAGACT T GAAT GGTAGTAGCCACT GGGGAAT CAT T GCAACT TATAGT GGAGCT GGCTAT TAT
CT GGAT T T
GT CAAGAACAAGAGAGGAAACAGCT GCACAAGT T GC TAGC C T CAAGAAAAAT GT CT GGCT GGAC C
GAG GAAC CA
GGGCAACT T T TAT T GACT T CT CAGT GTACAACGCCAACAT TAACCT GT T CT GT GT GGT
CAGGT TAT T GGT T GAA

T T CCCAGCAACAGGT GGT GT GAT T CCAT CT T GGCAAT T T CAGCCT T TAAAGCT GAT
CCGATAT GT CACAACT T T
T GAT T T CT T CCT GGCAGCCT GT GAGAT TAT CT T T T GT T T CT T TAT CT T T
TACTAT GT GGT GGAAGAGATAT T GG
AAAT T CGCAT T CACAAACTACACTAT T T CAGGAGT T T CT GGAAT T GT CT GGAT GT T GT
GAT CGT T GT GCT GT CA
GT GGTAGCTATAGGAAT TAACATATACAGAACAT CAAAT GT GGAGGT GCTAC TACAGT T T CT
GGAAGAT CAAAA
TACT T T CCCCAACT T T GAGCAT CT GGCATAT T GGCAGATACAGT T CAACAATATAGCT GCT GT
CACAGTAT T T T
TT GT CT GGAT TAAGCT CT T CAAAT T CAT CAAT T T TAACAGGACCAT GAGCCAGCT CT
CGACAACCAT GT CT CGA
T GT GCCAAAGACCT GT T T GGCT T T GCTAT TAT GT T CT T CAT TAT T T T CCTAGCGTAT
GCT CAGT T GGCATACCT
T GT CT T T GGCACT CAGGT CGAT GACT T CAGTACT T T CCAAGAGT GTAT CT T CACT CAAT
T CCGTAT CAT T T T GG
GCGATAT CAACT T T GCAGAGAT T GAGGAAGCTAAT CGAGT T T T GGGACCAAT T TAT T T
CACTACAT T T GT GT T C
T T TAT GT T CT T CAT T CT T T T GAATAT GT T T T T GGCTAT CAT CAAT GATACT TACT
CT GAAGT GAAAT CT GACT T
GGCACAGCAGAAAGCT GAAAT GGAACT CT CAGAT CT TAT CAGAAAGGGCTAC CATAAAGCT T T GGT
CAAAC TAA
AACT GAAAAAAAATACCGT GGAT GACAT T T CAGAGAGT CT GCGGCAAGGAGGAGGCAAGT TAAACT T
T GAC GAA
CT T CGACAAGAT CT CAAAGGGAAGGGCCATACT GAT GCAGAGAT T GAGGCAATAT T CACAAAGTAC
GAC CAAGA
TGGAGACCAAGAACTGACCGAACATGAACATCAGCAGATGAGAGACGACTTGGAGAAAGAGAGGGAGGACCTGG
AT T T GGAT CACAGT T CT T TACCACGT CCCAT GAGCAGCCGAAGT T T CCCT CGAAGCCT GGAT
GACT CT GAGGAG
GAT GAC GAT GAAGATAGCGGACATAGCT CCAGAAGGAGGGGAAGCAT T T CTAGT GGCGT T T CT TAC
GAAGAGT T
T CAAGT CCT GGT GAGACGAGT GGACCGGAT GGAGCAT T CCAT CGGCAGCATAGT GT CCAAGAT T
GACGCCGT GA
T CGT GAAGCTAGAGAT TAT GGAGCGAGCCAAACT GAAGAGGAGGGAGGT GCT GGGAAGGCT GT T GGAT
GGGGT G
GCCGAGGAT GAAAGGCT GGGT CGT GACAGT GAAAT CCATAGGGAACAGAT GGAACGGCTAGTAC GT
GAAGAGT T
GGAACGCT GGGAAT CCGAT GAT GCAGCT T CCCAGAT CAGT CAT GGT T TAGGCACGCCAGT
GGGACTAAAT GGT C
AACCT CGCCCCAGAAGCT CCCGCCCAT CT T CCT CCCAAT CTACAGAAGGCAT GGAAGGT GCAGGT
GGAAAT GGG
AGT T CTAAT GT CCACGTAT GA
SEQ ID NO:4 PC-2 polypeptide MVNS SRVQPQQPGDAKRP PAP RAP D P GRLMAGCAAVGAS LAAP GGLCEQRGLE I EMQRI RQAAARDP
PAGAAAS
P S P P L S S CS RQAWS RDNP GFEAEEEEEEVEGEEGGMVVEMDVEWRP GS RRSAAS SAVS
SVGARSRGLGGYHGAG
HP SGRRRRREDQGP PCPS PVGGGDPLHRHLPLEGQP PRVAWAERLVRGLRGLWGTRLMEES
STNREKYLKSVLR
ELVTYLL FL IVLC I LTYGMMS SNVYYYTRMMSQLFLDT PVS KT EKTNFKT L S
SMEDFWKFTEGSLLDGLYWKMQ
P SNQTEADNRS FI FYENLLLGVP RI RQLRVRNGSCS I PQDLRDE I KECYDVYSVS SEDRAP
FGPRNGTAWIYT S
EKDLNGS SHWGI IATYS GAGYYLDL S RT REETAAQVAS LKKNVWLDRGT RAT FI D FSVYNANINL
FCVVRLLVE
FPATGGVI P SWQ FQ P LKL I RYVTT FD FFLAACE I I FCFFI FYYVVEE I LEI RI HKLHYFRS
FWNCLDVVIVVLS
VVAI GIN I YRT SNVEVLLQFLEDQNT FPNFEHLAYWQ I Q ENNIAAVTVFFVWI KL FKFINFNRTMS
QL S T TMS R
CAKDL FGFAIMFFI I FLAYAQLAYLVFGTQVDD FS T FQEC I FTQ FRI I LGD INFAE I
EEANRVLGP I YFT T FVF
FMFFI LLNMFLAI INDTYS EVKS DLAQQKAEMEL S DL I RKGYHKALVKLKLKKNTVDD I SES
LRQGGGKLNFDE
LRQDLKGKGHT DAE I EAT FT KYDQDGDQELT EHEHQQMRDDLEKEREDLDLDH S SLP RPMS S RS FP
RS LDD S EE

DDDEDSGHS S RRRGS I S S GVS YEEFQVLVRRVDRMEHS I GS IVS KI
DAVIVKLEIMERAKLKRREVLGRLLDGV
AEDERLGRDSEIHREQMERLVREELERWESDDAASQI SHGLGTPVGLNGQPRPRS S RP S S
SQSTEGMEGAGGNG
S SNVHV
SEQ ID NO:5 HDAd-PK1)1 RightITR-CBh-mCherry:PKD1-HGHpA-PackagingSignal-LeftITR
CCCGTAGAAAAGATCAAAGGATCT TCTTGAGATCCT TT TT TICTGCGCGTAATCTGCTGCTTGCAAAC
AAAAAAACCACCGCTACCAGCGGIGGITTGITTGCCGGATCAAGAGCTACCAACTCTITTICCGAAGG
TAACTGGCTICAGCAGAGCGCAGATACCAAATACTGICCTICTAGIGTAGCCGTAGTTAGGCCACCAC
TICAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCIGTTACCAGTGGCTGCTGCCAG
TGGCGATAAGTCGTGICTTACCGGGITGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGICGG
GCTGAACGGGGGGITCGTGCACACAGCCCAGCTIGGAGCGAACGACCTACACCGAACTGAGATACCTA
CAGCGTGAGCTATGAGAAAGCGCCACGCTICCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG
CAGGGICGGAACAGGAGAGCGCACGAGGGAGCTICCAGGGGGAAACGCCIGGTATCTITATAGTCCTG
TCGGGT TTCGCCACCTCTGACT TGAGCGTCGATT TT TGTGATGCTCGTCAGGGGGGCGGAGCCTATGG
AAAAACGCCAGCAACGCGGCCT TT TTACGGITCCIGGCCT TT TGCTGGCCTT TTGCTCACATGT TCTT
TCCTGCGTTATCCCCTGATTCTGIGGATAACCGTATTACCGCCITTGAGTGAGCTGATACCGCTCGCC
GCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTT
CTCCTTACGCATCTGTGCGGTATT TCACACCGCATATGGATCCATGCATGTTAAGT T TAAACATCATC
AATAATATAC C T TATT TTGGAT TGAAGC CAATATGATAATGAGGGGGTGGAG TT TG TGAC GTGGCGC
G
GGGCGTGGGAACGGGGCGGGTGACGTAGGT TT TAGGGCGGAGTAACTIGTATGIGT TGGGAATTGTAG
TT TI CT TAAAATGGGAAGTTACGTAACGTGGGAAAACGGAAGTGACGATT TGAGGAAGTTGIGGGIT T
TT TGGCTT TCGT TICTGGGCGTAGGT TCGCGTGCGGTT TICTGGGIGT TT TT TGTGGACT TTAACCGT
TACGTCATTTTTTAGTCCTATATATACTCGCTCTGCACTTGGCCCTTTTTTACACTGTGACTGATTGA
GCTGGIGCCGTGICGAGTGGIGITTITTGATGCCCCCCCTCGAGGITCGACGGTATCGATAAGCTTGA
TT TAAT TAAGGCCGGCCCCTAGGGGCGCGCGCGGCCGCTAGGGATAACAGGGTAAT TGTTGACAATTA
ATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGA
CCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTC
GGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCAT
CAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACG
AGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACC
GAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTT
CGTGGCCGAGGAGCAGGACTGAACGCGTCGTTACATAACTTACGGTAAATGGCCCGCCIGGCTGACCG
CCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGITCCCATAGTAACGCCAATAGGGACTTT
CCATTGACGTCAATGGGIGGAGTATTTACGGTAAACTGCCCACTIGGCAGTACATCAAGIGTATCATA
TGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCIGGCATTATGCCCAGTACATG
ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGT
GAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTA

TT TT TTAATTAT TT TGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGG
CGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCG
AAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGG
GAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCT
CTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGC
TGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCAC
CTGICCGGAGAATT CGCCACCATGCCGCCCGCCGCGCCCGCCCGCCTGGCGCTGGCCCTGGGCCTGGG
_ CC TG TGGC TC GGGGCGC TGGCGGGGGGC CC CGGGATGG TGAGCAAGGGCGAGGAGGATAACATGGCCA
TCATCAAGGAGT TCATGC GC TTCAAGGTGCACATGGAGGGC TCC GTGAAC GGCCAC GAGT TC GAGATC
GAGGGC GAGGGC GAGGGC CGCC CC TACGAGGGCACC CAGACC GC CAAGC TGAAGGTGACCAAGGGTGG
CCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGC
ACCCCGCCGACATCCCCGAC TACT TGAAGC TGTCCT TCCCCGAGGGCT TCAAGTGGGAGCGCGTGATG
AAC T TC GAGGAC GGCGGC GTGG TGAC CG TGAC CCAGGAC TCC TC CC TGCAGGAC GGCGAG
TTCATC TA
CAAGGTGAAGC TGC GC GGCACCAAC T TC CC C TCC GACGGC CC CG TAATGCAGAAGAAGAC
CATGGGC T
GGGAGGCC TC C TCC GAGC GGATGTAC CC CGAGGACGGC GC CC TGAAGGGC GAGATCAAGCAGAGGC
TG
AAGC TGAAGGAC GGCGGC CAC TAC GACGC TGAGG TCAAGACCAC C TACAAGGCCAAGAAGCC CG
TGCA
GC TGCC CGGC GC C TACAACG TCAACATCAAGT TGGACATCAC C TCC CACAAC GAGGAC
TACACCATC G
TGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGGGCGCG
CCGGGGGGCCCCGGGCGCGGCTGCGGGCCCTGCGAGCCCCCCTGCCTCTGCGGCCCAGCGCCCGGCGC
CGCCTGCCGCGTCAACTGCTCGGGCCGCGGGCTGCGGACGCTCGGTCCCGCGCTGCGCATCCCCGCGG
ACGCCACAGCGCTAGACGTCTCCCACAACCTGCTCCGGGCGCTGGACGTTGGGCTCCTGGCGAACCTC
TC GGCGC TGGCAGAGC TGGATATAAGCAACAACAAGAT TTC TAC GT TAGAAGAAGGAATATT TGC TAA
TT TATT TAAT TTAAGTGAAATAAACC TGAG TGGGAACC CG TT TGAG TG TGAC TG TGGC C TGGCG
TGGC
TGCC GC GATGGGCGGAGGAGCAGCAGGTGC GGGTGG TGCAGC CC GAGGCAGC CACG TG TGC TGGGCC
T
GGCTCCCTGGCTGGCCAGCCTCTGCTTGGCATCCCCTTGCTGGACAGTGGCTGTGGTGAGGAGTATGT
CGCC TGCC TC CC TGACAACAGC TCAGGCAC CG TGGCAGCAGTGTCC TT TTCAGC TGCC CACGAAGGC
C
TGCTTCAGCCAGAGGCCTGCAGCGCCTTCTGCTTCTCCACCGGCCAGGGCCTCGCAGCCCTCTCGGAG
CAGGGCTGGTGCCTGTGTGGGGCGGCCCAGCCCTCCAGTGCCTCCTTTGCCTGCCTGTCCCTCTGCTC
CGGCCCCCCGCCACCTCCTGCCCCCACCTGTAGGGGCCCCACCCTCCTCCAGCACGTCTTCCCTGCCT
CCCCAGGGGCCACCCTGGTGGGGCCCCACGGACCTCTGGCCTCTGGCCAGCTAGCAGCCTTCCACATC
GC TGCCCCGC TCCC TGTCAC TGCCACACGC TGGGAC TTCGGAGACGGC TCCGCCGAGGTGGATGCCGC
TGGGCCGGCTGCCTCGCATCGCTATGTGCTGCCTGGGCGCTATCACGTGACGGCCGTGCTGGCCCTGG
GGGCCGGCTCAGCCCTGCTGGGGACAGACGTGCAGGTGGAAGCGGCACCTGCCGCCCTGGAGCTCGTG
TGCC CG TC C TCGGTGCAGAG TGAC GAGAGC C T TGAC C TCAGCATCCAGAACC GC GG TGGT
TCAGGCC T
GGAGGCCGCCTACAGCATCGTGGCCCTGGGCGAGGAGCCGGCCCGAGCGGTGCACCCGCTCTGCCCCT
CGGACACGGAGATCTTCCCTGGCAACGGGCACTGCTACCGCCTGGTGGTGGAGAAGGCGGCCTGGCTG
CAGGCGCAGGAGCAGTGTCAGGCC TGGGCC GGGGCC GC CC TGGCAATGGTGGACAG TC CC GC CG TGCA

GCGCTTCCTGGTCTCCCGGGTCACCAGGAGCCTAGACGTGTGGATCGGCTTCTCGACTGTGCAGGGGG
TGGAGG TGGGCC CAGC GC CGCAGGGC GAGGCC TTCAGC C TGGAGAGC TGC CAGAAC TGGC TGCC
CGGG
GAGCCACACCCAGCCACAGCCGAGCACTGCGTCCGGCTCGGGCCCACCGGGTGGTGTAACACCGACCT
GTGC TCAGCGCC GCACAGC TAC GTC TGC GAGC TGCAGC CC GGAGGC CCAG TGCAGGATGC
CGAGAAC C
TCCTCGTGGGAGCGCCCAGTGGGGACCTGCAGGGACCCCTGACGCCTCTGGCACAGCAGGACGGCCTC
TCAGCCCCGCACGAGCCCGTGGAGGTCATGGTATTCCCGGGCCTGCGTCTGAGCCGTGAAGCCTTCCT

CACCACGGCCGAATTTGGGACCCAGGAGCTCCGGCGGCCCGCCCAGCTGCGGCTGCAGGTGTACCGGC
TC C TCAGCACAGCAGGGACC CC GGAGAACGGCAGCGAGCC TGAGAGCAGG TC CC CGGACAACAGGAC C
CAGCTGGCCCCCGCGTGCATGCCAGGGGGACGCTGGTGCCCTGGAGCCAACATCTGCTTGCCGCTGGA
CGCCTCCTGCCACCCCCAGGCCTGCGCCAATGGCTGCACGTCAGGGCCAGGGCTACCCGGGGCCCCCT
ATGCGCTATGGAGAGAGTTCCTCTTCTCCGTTCCCGCGGGGCCCCCCGCGCAGTACTCGGTCACCCTC
CACGGCCAGGATGTCCTCATGCTCCCTGGTGACCTCGTTGGCTTGCAGCACGACGCTGGCCCTGGCGC
CC TCCTGCAC TGCTCGCCGGCTCCCGGCCACCCTGGTCCCCAGGCCCCGTACCTCTCCGCCAACGCC T
CGTCATGGCTGCCCCACTTGCCAGCCCAGCTGGAGGGCACTTGGGCCTGCCCTGCCTGTGCCCTGCGG
CTGCTTGCAGCCACGGAACAGCTCACCGTGCTGCTGGGCTTGAGGCCCAACCCTGGACTGCGGCTGCC
TGGGCGCTATGAGGTCCGGGCAGAGGTGGGCAATGGCGTGTCCAGGCACAACCTCTCCTGCAGCTTTG
ACGTGGTCTCCCCAGTGGCTGGGCTGCGGGTCATCTACCCTGCCCCCCGCGACGGCCGCCTCTACGTG
CCCACCAACGGCTCAGCCTTGGTGCTCCAGGTGGACTCTGGTGCCAACGCCACGGCCACGGCTCGCTG
GCCTGGGGGCAGTGTCAGCGCCCGCTTTGAGAATGTCTGCCCTGCCCTGGTGGCCACCTTCGTGCCCG
GC TGCC CC TGGGAGAC CAAC GATACC C TGT TC TCAG TGGTAGCAC TGC CG TGGC TCAG
TGAGGGGGAG
CACG TGGTGGAC GTGG TGGTGGAAAACAGC GC CAGC CGGGCCAACC TCAGCC TGCGGG TGAC GGCGGA

GGAGCCCATCTGTGGCCTCCGCGCCACGCCCAGCCCCGAGGCCCGTGTACTGCAGGGAGTCCTAGTGA
GG TACAGC CC CG TGGTGGAGGC CGGC TC GGACATGG TC TTCC GG TGGACCATCAAC
GACAAGCAGTC C
C TGACC TTCCAGAACG TGGTC T TCAATG TCAT TTATCAGAGC GC GGCGGTC T TCAAGC TC TCAC
TGAC
GGCCTCCAACCACGTGAGCAACGTCACCGTGAACTACAACGTAACCGTGGAGCGGATGAACAGGATGC
AGGGTCTGCAGGTCTCCACAGTGCCGGCCGTGCTGTCCCCCAATGCCACGCTAGCACTGACGGCGGGC
GTGCTGGTGGACTCGGCCGTGGAGGTGGCCTTCCTGTGGACCTTTGGGGATGGGGAGCAGGCCCTCCA
CCAGTTCCAGCCTCCGTACAACGAGTCCTTCCCGGTTCCAGACCCCTCGGTGGCCCAGGTGCTGGTGG
AGCACAATGTCATGCACACC TACGC TGC CC CAGG TGAG TACC TC C TGACC GTGC TGGCATC
TAATGC C
TTCGAGAACCTGACGCAGCAGGTGCCTGTGAGCGTGCGCGCCTCCCTGCCCTCCGTGGCTGTGGGTGT
GAGTGACGGCGTCCTGGTGGCCGGCCGGCCCGTCACCTTCTACCCGCACCCGCTGCCCTCGCCTGGGG
GTGTTCTTTACACGTGGGACTTCGGGGACGGCTCCCCTGTCCTGACCCAGAGCCAGCCGGCTGCCAAC
CACACC TATGCC TC GAGGGGCACC TACCAC GTGC GC C TGGAGGTCAACAACACGGTGAGC GG TGCGGC

GGCCCAGGCGGATGTGCGCGTCTTTGAGGAGCTCCGCGGACTCAGCGTGGACATGAGCCTGGCCGTGG
AGCAGGGCGCCCCCGTGGTGGTCAGCGCCGCGGTGCAGACGGGCGACAACATCACGTGGACCTTCGAC
ATGGGGGACGGCACCGTGCTGTCGGGCCCGGAGGCAACAGTGGAGCATGTGTACCTGCGGGCACAGAA
CTGCACAGTGACCGTGGGTGCGGCCAGCCCCGCCGGCCACCTGGCCCGGAGCCTGCACGTGCTGGTCT
TCGTCCTGGAGGTGCTGCGCGTTGAACCCGCCGCCTGCATCCCCACGCAGCCTGACGCGCGGCTCACG
GCCTACGTCACCGGGAACCCGGCCCACTACCTCTTCGACTGGACCTTCGGGGATGGCTCCTCCAACAC
GACCGTGCGGGGGTGCCCGACGGTGACACACAACTTCACGCGGAGCGGCACGTTCCCCCTGGCGCTGG
TGC TGTCCAGCC GC GTGAACAGGGCGCATTAC TTCACCAGCATC TGCG TGGAGC CAGAGG TGGGCAAC
GTCACC C TGCAGCCAGAGAGGCAG TT TG TGCAGC TC GGGGAC GAGGCC TGGC TGGTGGCATG TGCC
TG
GCCCCCGTTCCCCTACCGCTACACCTGGGACTTTGGCACCGAGGAAGCCGCCCCCACCCGTGCCAGGG
GC CC TGAGGTGACG TTCATC TACC GAGACC CAGGC TCC TATC TTGTGACAGTCACC GC
GTCCAACAAC
ATCTCTGCTGCCAATGACTCAGCCCTGGTGGAGGTGCAGGAGCCCGTGCTGGTCACCAGCATCAAGGT
CAATGGCTCCCTTGGGCTGGAGCTGCAGCAGCCGTACCTGTTCTCTGCTGTGGGCCGTGGGCGCCCCG
CCAGCTACCTGTGGGATCTGGGGGACGGTGGGTGGCTCGAGGGTCCGGAGGTCACCCACGCTTACAAC
AGCACAGG TGAC TTCACC GT TAGGGTGGCC GGC TGGAATGAGGTGAGC CGCAGC GAGGCC TGGC TCAA

TGTGACGGTGAAGCGGCGCGTGCGGGGGCTCGTCGTCAATGCAAGCCGCACGGTGGTGCCCCTGAATG

GGAGCG TGAGC T TCAGCACG TC GC TGGAGGCC GGCAGTGATG TGCGC TAT TC C TGGGTGC TC TG
TGAC
CGCTGCACGCCCATCCCTGGGGGTCCTACCATCTCTTACACCTTCCGCTCCGTGGGCACCTTCAATAT
CATCGTCACGGCTGAGAACGAGGTGGGCTCCGCCCAGGACAGCATCTTCGTCTATGTCCTGCAGCTCA
TAGAGGGGC TGCAGGTGG TGGGCGGTGGCC GC TAC T TC CC CACCAACCACAC GG TACAGC TGCAGGC
C
GTGG TTAGGGATGGCACCAACG TC TC C TACAGC TGGAC TGCC TGGAGGGACAGGGGCC CGGC CC
TGGC
CGGCAGCGGCAAAGGCTTCTCGCTCACCGTGCTCGAGGCCGGCACCTACCATGTGCAGCTGCGGGCCA
CCAACATGCTGGGCAGCGCCTGGGCCGACTGCACCATGGACTTCGTGGAGCCTGTGGGGTGGCTGATG
GTGGCCGCCTCCCCGAACCCAGCTGCCGTCAACACAAGCGTCACCCTCAGTGCCGAGCTGGCTGGTGG
CAGTGGTGTCGTATACACTTGGTCCTTGGAGGAGGGGCTGAGCTGGGAGACCTCCGAGCCATTTACCA
CCCATAGCTTCCCCACACCCGGCCTGCACTTGGTCACCATGACGGCAGGGAACCCGCTGGGCTCAGCC
AACGCCACCGTGGAAGTGGATGTGCAGGTGCCTGTGAGTGGCCTCAGCATCAGGGCCAGCGAGCCCGG
AGGCAGCT TCGTGGCGGCCGGGTCCTCTGTGCCC TT TTGGGGGCAGCTGGCCACGGGCACCAATGTGA
GC TGGTGC TGGGCTGTGCCCGGCGGCAGCAGCAAGCGTGGCCCTCATGTCACCATGGTCT TCCCGGAT
GC TGGCAC C T TC TC CATC CGGC TCAATGCC TC CAAC GCAG TCAGC TGGGTC TCAGC CACG
TACAACC T
CACGGCGGAGGAGCCCATCGTGGGCCTGGTGCTGTGGGCCAGCAGCAAGGTGGTGGCGCCCGGGCAGC
TGGTCCAT TT TCAGATCC TGCTGGCTGCCGGC TCAGCTGTCACC TTCCGCCTGCAGGTCGGCGGGGCC
AACCCCGAGGTGCTCCCCGGGCCCCGTTTCTCCCACAGCTTCCCCCGCGTCGGAGACCACGTGGTGAG
CGTGCGGGGCAAAAACCACGTGAGCTGGGCCCAGGCGCAGGTGCGCATCGTGGTGCTGGAGGCCGTGA
GTGGGC TGCAGG TGCC CAAC TGC TGC GAGC C TGGCATC GC CACGGGCAC TGAGAGGAAC T
TCACAGC C
CGCGTGCAGCGCGGCTCTCGGGTCGCCTACGCCTGGTACTTCTCGCTGCAGAAGGTCCAGGGCGACTC
GC TGGTCATCCTGTCGGGCCGCGACGTCACCTACACGCCCGTGGCCGCGGGGCTGT TGGAGATCCAGG
TGCGCGCCTTCAACGCCCTGGGCAGTGAGAACCGCACGCTGGTGCTGGAGGTTCAGGACGCCGTCCAG
TATGTGGCCCTGCAGAGCGGCCCCTGCTTCACCAACCGCTCGGCGCAGTTTGAGGCCGCCACCAGCCC
CAGC CC CC GGCG TG TGGC C TAC CAC TGGGAC T
TTGGGGATGGGTCGCCAGGGCAGGACACAGATGAGC
CCAGGGCCGAGCACTCCTACCTGAGGCCTGGGGACTACCGCGTGCAGGTGAACGCCTCCAACCTGGTG
AGCTTCTTCGTGGCGCAGGCCACGGTGACCGTCCAGGTGCTGGCCTGCCGGGAGCCGGAGGTGGACGT
GG TC C TGC CC C TGCAGGTGC TGATGC GGCGATCACAGC GCAAC TAC TTGGAGGC CCAC GT TGAC
C TGC
GC GAC TGC GTCACC TACCAGAC TGAG TACC GC TGGGAGGTGTATCGCACC GC CAGC TGCCAGCGGCC
G
GGGCGCCCAGCGCGTGTGGCCCTGCCCGGCGTGGACGTGAGCCGGCCTCGGCTGGTGCTGCCGCGGCT
GGCGCTGCCTGTGGGGCACTACTGCTTTGTGTTTGTCGTGTCATTTGGGGACACGCCACTGACACAGA
GCATCCAGGCCAATGTGACGGTGGCCCCCGAGCGCCTGGTGCCCATCATTGAGGGTGGCTCATACCGC
GTGTGGTCAGACACACGGGACCTGGTGCTGGATGGGAGCGAGTCCTACGACCCCAACCTGGAGGACGG
CGACCAGACGCCGCTCAGTTTCCACTGGGCCTGTGTGGCTTCGACACAGAGGGAGGCTGGCGGGTGTG
CGCTGAAC TT TGGGCCCCGCGGGAGCAGCACGGTCACCAT TCCACGGGAGCGGC TGGCGGCTGGCGTG
GAGTACACCTTCAGCCTGACCGTGTGGAAGGCCGGCCGCAAGGAGGAGGCCACCAACCAGACGGTGCT
GATCCGGAGTGGCCGGGTGCCCATTGTGTCCTTGGAGTGTGTGTCCTGCAAGGCACAGGCCGTGTACG
AAGTGAGC CGCAGC TC C TAC GTGTAC TTGGAGGGCC GC TGCC TCAATTGCAGCAGC GGC
TCCAAGCGA
GGGC GG TGGGC TGCAC GTAC GT TCAGCAACAAGACGC TGG TGC TGGATGAGACCAC CACATC
CACGGG
CAGTGCAGGCATGCGACTGGTGCTGCGGCGGGGCGTGCTGCGGGACGGCGAGGGATACACCTTCACGC
TCACGGTGCTGGGCCGCTCTGGCGAGGAGGAGGGCTGCGCCTCCATCCGCCTGTCCCCCAACCGCCCG
CCGCTGGGGGGCTCTTGCCGCCTCTTCCCACTGGGCGCTGTGCACGCCCTCACCACCAAGGTGCACTT
CGAATGCACGGGCTGGCATGACGCGGAGGATGCTGGCGCCCCGCTGGTGTACGCCCTGCTGCTGCGGC
GC TG TC GC CAGGGC CAC TGC GAGGAG TTC TGTGTC TACAAGGGCAGCC TC TC CAGC TACGGAGC
CGTG

CTGCCCCCGGGTTTCAGGCCACACTTCGAGGTGGGCCTGGCCGTGGTGGTGCAGGACCAGCTGGGAGC
CGC TGTGG TC GC CC TCAACAGG TC TT TGGC CATCAC CC TC CCAGAGCC CAAC GGCAGC
GCAACGGGGC
TCACAGTCTGGCTGCACGGGCTCACCGCTAGTGTGCTCCCAGGGCTGCTGCGGCAGGCCGATCCCCAG
CACGTCATCGAGTACTCGTTGGCCCTGGTCACCGTGCTGAACGAGTACGAGCGGGCCCTGGACGTGGC
GGCAGAGCCCAAGCACGAGCGGCAGCACCGAGCCCAGATACGCAAGAACATCACGGAGACTCTGGTGT
CC C TGAGGGTCCACAC TG TGGATGACATCCAGCAGATC GC TGC TGC GC TGGC CCAG TGCATGGGGCC
C
AGCAGGGAGC TC GTATGC CGC TCG TGCC TGAAGCAGAC GC TGCACAAGC TGGAGGC CATGATGC
TCAT
CC TGCAGGCAGAGACCAC CGCGGGCACC GTGACGCC CACC GC CATC GGAGACAGCATC C TCAACATCA
CAGGAGAC C TCATC CACC TGGC CAGC TC GGAC GTGC GGGCAC CACAGC CC TCAGAGC TGGGAGC
CGAG
TCACCATCTCGGATGGTGGCGTCCCAGGCCTACAACCTGACCTCTGCCCTCATGCGCATCCTCATGCG
CTCCCGCGTGCTCAACGAGGAGCCCCTGACGCTGGCGGGCGAGGAGATCGTGGCCCAGGGCAAGCGCT
CGGACCCGCGGAGCCTGCTGTGCTATGGCGGCGCCCCAGGGCCTGGCTGCCACTTCTCCATCCCCGAG
GC TT TCAGCGGGGCCC TGGCCAACCTCAGTGACGTGGTGCAGCTCATC TT TC TGGTGGAC TCCAATCC
CTTTCCCTTTGGCTATATCAGCAACTACACCGTCTCCACCAAGGTGGCCTCGATGGCATTCCAGACAC
AGGC CGGC GC CCAGATCC CCATCGAGCGGC TGGC C TCAGAGC GC GC CATCAC CG TGAAGG TGCC
CAAC
AACTCGGACTGGGCTGCCCGGGGCCACCGCAGCTCCGCCAACTCCGCCAACTCCGTTGTGGTCCAGCC
CCAGGCCTCCGTCGGTGCTGTGGTCACCCTGGACAGCAGCAACCCTGCGGCCGGGCTGCATCTGCAGC
TCAAC TATAC GC TGC TGGAC GGCCAC TACC TG TC TGAGGAAC C TGAGC CC TACC TGGCAG TC
TACC TA
CAC TCGGAGC CC CGGC CCAATGAGCACAAC TGC TCGGC TAGCAGGAGGATCC GC CCAGAG TCAC
TCCA
GGGTGCTGACCACCGGCCCTACACCTTCTTCATTTCCCCGGGGAGCAGAGACCCAGCGGGGAGTTACC
ATCTGAACCTCTCCAGCCACTTCCGCTGGTCGGCGCTGCAGGTGTCCGTGGGCCTGTACACGTCCCTG
TGCCAG TAC T TCAGCGAGGAGGACATGG TG TGGC GGACAGAGGGGC TGC TGC CC C TGGAGGAGACC
TC
GCCCCGCCAGGCCGTCTGCCTCACCCGCCACCTCACCGCCTTCGGCGCCAGCCTCTTCGTGCCCCCAA
GC CATG TC CGC T TTGTGT TTCC TGAGCC GACAGC GGATGTAAAC TACATC GTCATGC TGACATG
TGC T
GTGTGC C TGG TGAC C TACATGG TCATGGCC GC CATC C TGCACAAGC TGGACCAG TTGGATGC
CAGCC G
GGGCCGCGCCATCCCTTTCTGTGGGCAGCGGGGCCGCTTCAAGTACGAGATCCTCGTCAAGACAGGCT
GGGGCCGGGGCTCAGGTACCACGGCCCACGTGGGCATCATGCTGTATGGGGTGGACAGCCGGAGCGGC
CACC GGCACC TGGACGGC GACAGAGC C T TC CACC GCAACAGC C TGGACATC T TC CGGATC GC
CACCC C
GCACAGCC TGGG TAGC GTGTGGAAGATC CGAG TG TGGCAC GACAACAAAGGGC TCAGC CC TGCC TGG
T
TC C TGCAGCACG TCATCG TCAGGGAC C TGCAGAC GGCACGCAGC GC C T TC TTCC TGGTCAATGAC
TGG
C T TTCGGTGGAGAC GGAGGC CAAC GGGGGC C TGG TGGAGAAGGAGG TGC TGGCC GC GAGC
GACGCAGC
CC TT TTGCGC TTCCGGCGCC TGCTGGTGGC TGAGCTGCAGCGTGGC TTCT TTGACAAGCACATC TGGC
TCTCCATATGGGACCGGCCGCCTCGTAGCCGTTTCACTCGCATCCAGAGGGCCACCTGCTGCGTTCTC
CTCATCTGCCTCTTCCTGGGCGCCAACGCCGTGTGGTACGGGGCTGTTGGCGACTCTGCCTACAGCAC
GGGGCATGTGTCCAGGCTGAGCCCGCTGAGCGTCGACACAGTCGCTGTTGGCCTGGTGTCCAGCGTGG
TTGTCTATCCCGTC TACC TGGCCATCCT TT TTCTCT TCCGGATGTCCCGGAGCAAGGTGGCTGGGAGC
CC GAGC CC CACACC TGCC GGGCAGCAGG TGC TGGACATCGACAGC TGC C TGGAC TC GTCC GTGC
TGGA
CAGCTCCTTCCTCACGTTCTCAGGCCTCCACGCTGAGCAGGCCTTTGTTGGACAGATGAAGAGTGACT
TG TT TC TGGATGAT TC TAAGAG TC TGGTGTGC TGGC CC TC CGGC GAGGGAAC GC TCAG
TTGGCC GGAC
C TGC TCAG TGAC CC GTCCAT TG TGGG TAGCAATC TGCGGCAGC TGGCACGGGGC
CAGGCGGGCCATGG
GC TGGGCCCAGAGGAGGACGGC TTCTCCCTGGCCAGCCCC TACTCGCC TGCCAAATCC TTCTCAGCAT
CAGATGAAGACC TGATCCAGCAGG TC C T TGCC GAGGGGGTCAGCAGCC CAGC CC C TAC CCAAGACAC
C
CACATGGAAACGGACCTGCTCAGCAGCCTGTCCAGCACTCCTGGGGAGAAGACAGAGACGCTGGCGCT

GCAGAGGCTGGGGGAGCTGGGGCCACCCAGCCCAGGCCTGAACTGGGAACAGCCCCAGGCAGCGAGGC
TGTCCAGGACAGGACTGGTGGAGGGTCTGCGGAAGCGCCTGCTGCCGGCCTGGTGTGCCTCCCTGGCC
CACGGGCTCAGCCTGCTCCTGGTGGCTGTGGCTGTGGCTGTCTCAGGGTGGGTGGGTGCGAGCTTCCC
CCCGGGCGTGAGTGTTGCGTGGCTCCTGTCCAGCAGCGCCAGCTTCCTGGCCTCATTCCTCGGCTGGG
AGCCACTGAAGGTCTTGCTGGAAGCCCTGTACTTCTCACTGGTGGCCAAGCGGCTGCACCCGGATGAA
GATGACACCCTGGTAGAGAGCCCGGCTGTGACGCCTGTGAGCGCACGTGTGCCCCGCGTACGGCCACC
CCAC GGC T TTGCAC TC TTCC TGGC CAAGGAAGAAGC CC GCAAGG TCAAGAGGC TACATGGCATGC
TGC
GGAGCCTCCTGGTGTACATGCTTTTTCTGCTGGTGACCCTGCTGGCCAGCTATGGGGATGCCTCATGC
CATGGGCACGCCTACCGTCTGCAAAGCGCCATCAAGCAGGAGCTGCACAGCCGGGCCTTCCTGGCCAT
CACGCGGTCTGAGGAGCTCTGGCCATGGATGGCCCACGTGCTGCTGCCCTACGTCCACGGGAACCAGT
CCAGCC CAGAGC TGGGGC CC CCAC GGC TGC GGCAGG TGCGGC TGCAGGAAGCAC TC TACC
CAGACCC T
CCCGGCCCCAGGGTCCACACGTGCTCGGCCGCAGGAGGCTTCAGCACCAGCGATTACGACGTTGGCTG
GGAGAG TC C TCACAATGGC TCGGGGACG TGGGCC TATTCAGC GC CGGATC TGC TGGGGGCATGG TCC
T
GGGGCTCCTGTGCCGTGTATGACAGCGGGGGCTACGTGCAGGAGCTGGGCCTGAGCCTGGAGGAGAGC
CGCGACCGGCTGCGCTTCCTGCAGCTGCACAACTGGCTGGACAACAGGAGCCGCGCTGTGTTCCTGGA
GC TCACGCGC TACAGCCCGGCCGTGGGGCTGCACGCCGCCGTCACGCTGCGCCTCGAGTTCCCGGCGG
CCGGCCGCGCCCTGGCCGCCCTCAGCGTCCGCCCCTTTGCGCTGCGCCGCCTCAGCGCGGGCCTCTCG
CTGCCTCTGCTCACCTCGGTGTGCCTGCTGCTGTTCGCCGTGCACTTCGCCGTGGCCGAGGCCCGTAC
TTGGCACAGGGAAGGGCGCTGGCGCGTGCTGCGGCTCGGAGCCTGGGCGCGGTGGCTGCTGGTGGCGC
TGACGGCGGCCACGGCACTGGTACGCCTCGCCCAGCTGGGTGCCGCTGACCGCCAGTGGACCCGTTTC
GTGCGCGGCCGCCCGCGCCGCTTCACTAGCTTCGACCAGGTGGCGCAGCTGAGCTCCGCAGCCCGTGG
CC TGGCGGCC TCGC TGCTCTTCCTGC TTTTGGTCAAGGCTGCCCAGCAGC TACGCTTCGTGCGCCAGT
GGTCCGTCTTTGGCAAGACATTATGCCGAGCTCTGCCAGAGCTCCTGGGGGTCACCTTGGGCCTGGTG
GTGCTCGGGGTAGCCTACGCCCAGCTGGCCATCCTGCTCGTGTCTTCCTGTGTGGACTCCCTCTGGAG
CGTGGCCCAGGCCCTGTTGGTGCTGTGCCCTGGGACTGGGCTCTCTACCCTGTGTCCTGCCGAGTCCT
GGCACCTGTCACCCCTGCTGTGTGTGGGGCTCTGGGCACTGCGGCTGTGGGGCGCCCTACGGCTGGGG
GC TGTTATTC TCCGCTGGCGCTACCACGCC TTGCGTGGAGAGCTGTACCGGCCGGCCTGGGAGCCCCA
GGAC TACGAGATGG TGGAGT TG TTCC TGCGCAGGC TGC GC C TC TGGATGGGC C TCAGCAAGG
TCAAGG
AGTTCCGCCACAAAGTCCGCTTTGAAGGGATGGAGCCGCTGCCCTCTCGCTCCTCCAGGGGCTCCAAG
GTATCCCCGGATGTGCCCCCACCCAGCGCTGGCTCCGATGCCTCGCACCCCTCCACCTCCTCCAGCCA
GC TGGATGGGCTGAGCGTGAGCCTGGGCCGGC TGGGGACAAGGTGTGAGCCTGAGCCC TCCCGCCTCC
AAGC CG TG TTCGAGGC CC TGC TCACC CAGT TTGACC GAC TCAAC CAGGCCACAGAGGACG TC
TACCAG
CTGGAGCAGCAGCTGCACAGCCTGCAAGGCCGCAGGAGCAGCCGGGCGCCCGCCGGATCTTCCCGTGG
CCCATCCCCGGGCCTGCGGCCAGCACTGCCCAGCCGCCTTGCCCGGGCCAGTCGGGGTGTGGACCTGG
CCAC TGGC CC CAGCAGGACACC CC TTCGGGCCAAGAACAAGG TC CACC CCAGCAGCAC TTAGTCCTCC
TICCIGGCGGGGGIGGGCCGTGGAGTCGGAGTGGACACCGCTCAGTAT TACT TICTGCCGCTGICAAG
GCCGAGGGCCAGGCAGAATGGCTGCACGTAGGITCCCCAGAGAGCAGGCAGGGGCATCTGICTGICTG
TGGGCTICAGCACTITAAAGAGGCTGIGTGGCCAACCAGGACCCAGGGICCCCTCCCCAGCTCCCITG
GGAAGGACACAGCAGTATTGGACGGITTCTAGCCICTGAGATGCTAATTTATTICCCCGAGTCCTCAG
GTACAGCGGGCTGTGCCCGGCCCCACCCCCIGGGCAGATGICCCCCACTGCTAAGGCTGCTGGCTICA
GGGAGGGITAGCCTGCACCGCCGCCACCCTGCCCCTAAGTTATTACCICTCCAGTTCCTACCGTACTC
CCTGCACCGICTCACTGIGTGICTCGTGICAGTAAT TTATATGGIGTTAAAATGIGTATATT TT TGTA
TGTCACTATTTTCACTAGGGCTGAGGGGCCTGCGCCCAGAGCTGGCCTCCCCCAACACCTGCTGCGCT

TGGTAGGTGTGGTGGCGTTATGGCAGCCCGGCTGCTGCTTGGATGCGAGCTTGGCCTTGGGCCGGTGC
TGGGGGCACAGCTGTCTGCCAGGCACTCTCATCACCCCAGAGGCCTTGTCATCCTCCCTTGCCCCAGG
CCAGGTAGCAAGAGAGCAGCGCCCAGGCCTGCTGGCATCAGGTCTGGGCAAGTAGCAGGACTAGGCAT
GTCAGAGGACCCCAGGGTGGTTAGAGGAAAAGACTCCTCCTGGGGGCTGGCTCCCAGGGTGGAGGAAG
GT GACT GT GT GT GT GT GT GT GT GCGCGCGCGCACGCGCGAGT GT
GCTGTATGGCCCAGGCAGCCTCAA
GGCCCTCGGAGCTGGCTGTGCCTGCTTCTGTGTACCACTTCTGTGGGCATGGCCGCTTCTAGAACGGG
TGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAG
CCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGT
GGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGG
GAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGAT
TCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGT
TT TT TT GGTAGAGACGGGGT TTCACCATAT TGGCCAGGCT GGTCTCCAACTCCTAATCTCAGGT GATC
TACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTTA
ACTATAACGGTCCTAAGGTAGCGAAGTCGACCGAATCGTTGTCCCTTGTCACAGCCATTGAGAATTTT
GGCAGGGAGCAT GT TCTTAGAGCATT TT TAGGCTCT GCGGGACATAACAGCTCT GCCTCAGAGCACAT
GCCT TTCTCAGCTCCT GAAAGCCACT GATCAAAT TGGAACAT TT TGTACCTTAGGGAT GAGGATATCA
ACTCTCCCAGCCACTTAGAGGGATAAAT GT GATGAT GCAT TCAATT GT GACTACATCT GATCCCAACT
GTTGCTTCAGCTGCTCTCCTATAGCACATGGCGGGAGGCGTGCATCCCAGTAGCTACCTCCCCACTTT
TGGGGAGATGTGGTTCCATCCATGAAACCTGGGTACCCGCCTACCAGGTCCTGGCCTATCAGGTGGCA
GGGTCT GGTCAAAGAAGGGCAT GT GT GGTCTTCAGCAAGGGAGACAGGACGGTGGT GCAGAGCGTCTA
GACCCTCAGGGCAAGTCTCCCCCACACCTGCTCCCGGGGCAGTT GT CT TT GT GACCTCCCATCCCCCT
CT GT TTCATCCTCTATAAAATGAGGGGCTGAGCCCCAAAATAACAGGCTTCT TT GCCATGAT GCAAAA
CT GCTGAATCTT TCTT TCTGACACACAAGGCATCGAGCAGCCTCTGAAAGAACCAAAGCCACTAGCAG
GCTTCCTGACTT GGGT TT GTAGGTACTGAATACTCCCT TGAAAAATAAAAACATAGAGGCACTT TTCT
CCTGGCTGTTTATTACAGAACGAAGAAAAAACACACTGGCTTGAAACAGACGCCAGATTTCAAATGTA
GAGGT GAAATACGAGGT GGCAAT TAAAAT GT GAT TACAGAAAGT CT GGACACTGAGAAAAGT TTACAG
GACAGT GGGT GT GGGT TT TCTATAACAGACACTTAAATATACAT GACGATAATT GCAGATAGAAACCA
TCAAAGACAAACCCCAAATCAACTAATAAT GT TTACAGAT GT TCCCCCCCAAACCACAGAGCCT TACA
TCAAAACAAATACTGAAAGGCTTTAAACCAGGAACAGCTCGCCTTAACCCCACGAGGGTGCACACAAG
CTGGGCTTTTTCTCTCGGTCTGAATGGTAAAGGGAGGAGGATACTCTAGCTCCTCCAGGTGGATTGCT
GAGACAGGGCTCGGCTCACACACTGTCTCTGCGCCTCTCCCAAATCTGGAGAACTCTCCCAGCCTCCT
GGTAAAGT GTCTCT GT GGGGCACT TAACGATAAAACAGCT TCTGCT GTAAAGCTCATTAGGAAAGAGC
TAGCGGAGACTGAAAGGTTCGCAAAAGAGATTAAGAATCACACAAGGCAATAGGATTTTTAGTGAACA
TAGAAATAAATGGCCAAGTGGT TT TCTATT TGGCAT TT GTCAACTT GCACAACAACTCTT GGTCATAT
CCACATTGCTCATTGCATTAAAACCATAAGCGACTCAGCCACCTAGCTTAACAAGGTATCACTGGAGC
AAACAACACGGTCT GCATAT TT GTAACATT GTATAATAAACACAAAACAATGCATAGTAAACACAACT
CTACTGAAACAAAAGCCGTCGCTTTATTTACAAAGTCACAAAATGAAGTATAAATACTTCTGTCATTA
AT GTTTAGGAAAACCATTTACAAAATTTTCAAATAT GTACACGTAGCTTGAAAAATCACCAGCTTTCC
AT TT TGTCACAGGTAGAGAGAGGGATAAGCAT GGGCTGACAACACCACTCAAAT TGTAACGGGAGACA
ACTGCGGGTATGGATCGACACCACTTCCTAGAGTGATGTCACCATGGGGGTTTCTATGGGCATCCTGC
TCAGATTTAAAGTGCCCCAGCATCCTGGGTGACTTGCCCAGAATTCTGGGCTGTGGCATTTTGAGCAG
CAGCATGCTGTTCCAAAATGTCGTCGATCAGCCTCAAGTTGCACACCCAGTCTTCATCTGGGCTCACA
CAGGAGCCTTTCAAGAGAGCTTCAATGAAATCTACCTCATTGCAGTCAGGTGACGAAATCAGATCATT

TAGIGGGGGITGGGGCTGGCGCAAAAAGTCGGCAGGIGGCAGCTCAGGGGGAATATCCGTICTGICGA
ACGGACCTGGGAACTGGCTGGCAGCAACGGCAGAAGCAGCAGCAGCGGTGGCAGCAGCAGCCACATAG
CTIGGIGGCTCGATGCCCIGTATGGGGCTCAGGGGACTAAAGCTGGCCATACCCTGCTGGAGGAACTT
GGTGGTGTTTGCTACAGGCACCGGGCCCTGTACCGGGCTCTGCCTGAGGCTCTGGCTGCCCAGCAGGC
TGAAGCTGGGGITGTTGGCCAGGGGCACTIGTGITCCCATCGCAGCGGGCACTIGTGCCTCCCAATCA
GATGGCCICTGAAGGCAGGCCIGGCCAGAAGGTGAGTGCTGCTGAACGCTATTATCCACTIGGCTGAG
GGGIGTITTCCCCGAAACTGCTGIGGICACAGCTGCTGCCGCTGTGACCCATGCAGCATTGTTGAACG
CAGTGGGCATTCTTGGCACACTAGGCCGTCTGAGCTGGTGGGGACTCAAGGACTGGGTGCCCAGGGAG
CTGGGACAGAACCCAGGCAGGGGCACTTCTGGTGGGGTGGCCTTGGGGCTCTGCATATGCTGGCAGAC
AGAGICAAGICTGCCCAGGGGAGICTGGCCTGAGIGTGAGAGGATGGGACACTGGGGGCTGGAGGTGA
AAATTCCITGCCGCTICCCCAGAGTTGGTGAGATCACTCCCATGCCCTCGCAGCTCTGGIGCCTGGIG
AGIGGGATCATTCCIGGACTCAGATTGITCTGAAGAAGCCCAGTTCTGGGIGGCATCAAGTGCTTGCT
AGATGGGGGGCTTGCCTTGATCCGGCTACACTTGGAGGTGACTTGTTCTTGGACGGCTACATACAGAA
AGAGAGAAGIGGGGATGAGTTCCAAAGGCATCCTCGACTICGGCTGIGGCCACCGGAGGGTAGCTCCT
GGCCCAACACGGACTICTCACCTCCCGCCCTIGGCTCTCTACTGAGCTCCCCCCTGCTCCCCAATTCC
TCGCCATTCCCCTCAT TICTCTGCCCTCAGCCIGGACTGCAGTTCT TCTGGGAAGCTGCCCCAACTCC
CTAGGICTGTGCTCACCAAGAGCAGATCACACTGGACTGAAATGCCAGCTGATTTGICTCTICAAGAA
AATTGGAAGCTCCIGGAGGICAGGGICCATGICTGCTITTACACTCAGTGCTCTGTATGCAGGCCIGG
CACTGCCCACCCITTGACAGGIGGIGCATATTITGTAGAAGGAAGGAAGGGGCCAGGIGGGGIGGGCT
GGGCTGGTGGCGGGAGCTAGCTCAGCCTCTTAGATTCTCTACCCGATGGATGTGACCTGGGACAGCAA
GTGAGTGTGGTGAGTGAGTGCAGACGGTGCTTTGTTCCCCTCTTGTCTCATAGCCTAGATGGCCTCTG
AGCCCAGATCTGGGGCTCAGACAACATTIGTICAACTGAACGGTAATGGGITTCCITTCTGAAGGCTG
AAATCTGGGAGCTGACAT TCTGGACTCCCTGAGT TCTGAAGAGCCTGGGGATGGAGAGACACGGAGCA
GAAGATGGAAGGTAGAGTCCCAGGIGCCTAAGATGGGGAATACATCTCCCCTCATTGICATGAGAGTC
CACTCTAGCTGATATCTACTGIGGCCAATATCTACCGGTACTITITTGGGGIGGACACTGAGICATGC
AGCAGICTTATGGITTACCCAAGGICAGGTAGGGGAGACAGTGCAGICAGAGCACAAGCCCAGTGIGT
CTGACCCACCCAAGAATCCATGCTCGTATCTACAAAAATGAT TT TT TCTCTIGTAATGGIGCCTAGGT
TCTT TTAT TATCATGGCATGIGTATGIT TT TCAACTAGGT TACAATCTGGCCITATAAGGITAACCTC
CIGGAGGCCACCAGCCTICCTGAAACTTGICTGTGCTGICCCTGCAACTGGAGTGTGCCTGATGTGGC
ACTCCAGCCIGGACAAGIGGGACACAGACTCCGCTGTTATCAGGCCCAAAGATGICTICCATAAGACC
AGAAGAGCAATGGIGTAGAGGIGICATGGGCTACAATAAAGATGCTGACCTCCTGICTGAGGGCAAGC
AGCCICTICTGGCCCTCAGACAAATGCTGAGTGITCCCAAGACTACCCTCGGCCTGGICCAATCTCAT
CCCACTGGIGCGTAAGGGITGCTGAACTCATGACTICTIGGCTAGCCTGCAACCTCCACGGAGTGGGA
ACTACATCAGGCAT TT TGCTAACTGCTGTATCCTAGGCCAATAAATGT TGATCACATT TATAGCTGCC
ATGGTAGGGIGGGGACCCCTGCTATCTATCTGIGGAGGCTCTGGGAGCCCCTGACACAAACTITCTGA
AGCAGAGCCTCCCCAACCCCTT TTCCAT TCCCTATACCTGACAGATGGCCCAGGAACCCATTAGAAAT
GGAAGGICACTGCAGCAGTATGTGAATGTGCGTGIGGGAGAAGGGCAGGATCAGAGCCCIGGGGGIGT
GGCAGCCCCCAAGTGATTCTAATCCAGATCCTAGGGITGITTCCCTGICCCATTGAAATAGCTGCTIT
AAGGGGCCTGACTCAGGGAAATCAGTCTCT TGAATTAAGTGGTGAT TT TGGAGTCATT TAGACCAGGC
CTICAATTGGGATCCACTAGTICTAGAGCGGCCGGGCCCAGGGAACCCCGCAGGCGGGGGCGGCCAGT
TTCCCGGGITCGGCTITACGTCACGCGAGGGCGGCAGGGAGGACGGAATGGCGGGGITTGGGGIGGGT
CCCTCCTCGGGGGAGCCCIGGGAAAAGAGGACTGCGTGIGGGAAGAGAAGGIGGAAATGGCGTITTGG
TTGACATGTGCCGCCTGCGAGCGTGCTGCGGGGAGGGGCCGAGGGCAGATTCGGGAATGATGGCGCGG

GGTGGGGGCGTGGGGGCT TTCTCGGGAGAGGCCCTTCCCT GGAAGT TT GGGGTGCGAT GGTGAGGTTC
TCGGGGCACCTCTGGAGGGGCCTCGGCACGGAAAGCGACCACCT GGGAGGGCGT GT GGGGACCAGGT T
TT GCCT TTAGTT TT GCACACACTGTAGT TCATCT TTAT GGAGAT GCTCAT GGCCTCAT TGAAGCCCCA

CTACAGCTCTGGTAGCGGTAACCATGCGTATTTGACACACGAAGGAACTAGGGAAAAGGCATTAGGTC
.. AT TTCAAGCCGAAATTCACATGTGCTAGAATCCAGATTCCAT GCTGACCGAT GCCCCAGGATATAGAA
AATGAGAATCTGGTCCTTACCTTCAAGAACATTCTTAACCGTAATCAGCCTCTGGTATCTTAGCTCCA
CCCTCACT GGTT TT TI CT TGTT TGTT GAACCGGCCAAGCT GCTGGCCTCCCTCCTCAACCGT TCTGAT
CATGCT TGCTAAAATAGTCAAAACCCCGGCCAGT TAAATATGCT TTAGCCTGCT TTAT TATGAT TAT T
TT TGTT GT TT TGGCAATGACCT GGTTACCT GT TGTT TCTCCCACTAAAACTT TT TAAGGGCAGGAATC
ACCGCCGTAACTCTAGCACTTAGCACAGTACTTGGCTTGTAAGAGGTCCTCGATGATGGTTTGTTGAA
TGAATACATTAAATAATTAACCACTT GAAC CC TAAGAAAGAAGC GAT T CT AT TT CATATTAGGCATT G

TAATGACTTAAGGTAAAGAGCAGTGCTATTAACGGAGTCTAACTGGGAATCCAGCTTGTTTGGGCTAT
TTACTAGTTGTGTGGCTGTGGGCAACTTACTTCACCTCTCTGGGCTTAAGTCATTTTATGTATATCTG
AGGT GCTGGCTACCTCTT GGAGTTAT TGAGAGGATTATAAGACAGTCTAT GT GAATCAGCAACCCTT G
CATGGCCCCT GGCGGGGAACAGTAATAATAGCCATCATCATGTT TACT TACATAGTCCTAAT TAGTCT
TCAAAACAGCCCTGTAGCAATGGTAT GATTAT TACCAT TT TACAGATGAGGAACCT TT GAAGCCTCAG
AGAGGCTAACAGACATACCCTAGGTCATACAGTTATTAAGAGAAGGAGCTCTGTCTCGAACCTAGCTC
TCTCTCTCTCGAGTAATACCAGTTAAAAAATAGGCTACAAATAGGTACTCAAAAAAATGGTAGTGGCT
GT TGTT TT TATTCAGT TGCT GAGGAAAAAATGTT GATT TT TCATCTCTAAACATCAACTTACTTAAT T
CTGCCAATTTCTTTTTTTTGAGACAGGGTCTCACTCTGTCACCTAGGATGGAGTGCAGTGGCACAATC
ACTGCTCACTGCAGCCTCGACTTCCCGGGCTCGGGTGATTCTCCCCAGGCTCAGGGGATTCTCCCACT
TCAGCCTCCCAAGTAGCTGGGACTACAGGTGCGCACCACCATCCCTGGCTAATATTTGTACTTTATTT
TATT TATT TATT TATT TATT TT TT GAGATGGAGT TTCGCTCT TGTT GCCCAAAT GAAT
TGCCTCTTAT
TTAATTTCGTCTGATGATACATTTTGTTTTTATTTTGTAAAAAATTATTTTTTTTCTTTTTGGAGACA
GGGTCTTGCTCTGTTGCCCAGGCTGGTCACAAACTCCTGACCTCAAGCAATCCTCCTGCCTTAGCCTC
CCAAAATGCT GGGATTACAGGCGT GACGACCTCGCCCGGCCT TGTATTAT GATACATT TT GAACAACT
ACAAGTAGACTTGGTATAATGAACCTGCACGTACCCATTGCCAAGTTCTGACAACTGTCTGTCTATAG
CCAATTAT GCAT TTCT TAAATTAGAACCCCCCCAATATACCCAAATATATATATAT GT GT GCATATAT
ATAGTAAGTT GTAACAAAGT TGTGAATTCATACCTGAAGTATCTCAAGTGAT GCAAGT TT TATGAAT T
.. TT TGTT TATGCCTT TT GGGAAGAGTT GTAT TGACAAAT TT TT TATGCT
TAAAGTAAACCATAAATCAA
AAAAATAAAATCTAGGATGCAATAAAACAAAACAACTTCTTGACATAAGTATGGTATGTAAATCTGTT
TT GATT GGAAATCAAT TT GT TATATT GCCAGAAT TCCT GT TT TAGAATACATCTCT GCTGATCT
GTCT
GTATTCTTAGACTGCATATCTGGGATGAACTCTGGGCAGAATTCACATGGGCTTCCTTTGAAATAAAC
AAGACT TT TCAAAT TCTTAGTCGATCTGCAGAACCT GTAGCCAGGCACTGAACCAT TT TGATAGATGC
.. AGTAATCGTT GCAAGT GTATAT TTCAAGGGAGTTCT GGCT GGGTCCTAGT TTAT GCTT GT
GGCAGAAG
CAGT GAGTAACT GGGAGGAAGT TGGT GAGTAAGCTTCAAGGAAGAAGTCATT TT TAGTACTCTGGATC
TTCCTGAT TT TAAAGCACTACAAAAT GGTGCATT TTCATTCT TGTCAAGT GATAACAGATATAT TCT G
AT GAGCCT GAAATGAATATATATT GTATCATT TT TATAATATCTAGCAAGGT TT GTAT TT TCCTAGAA
CT TGAACTAAAT TTCAGT TCATAAAATT TATAAAATACTTAGTT GT TGTAAAATAT TT TT GGAATGT T
CACATAGGTGACACACAAATGTCCCATTTTCATTCTTTCTATAGTAAATATGTTCTGATATGTGAAGG
TT TAGCAGAT GCATCAGCAT TTAATCCTAGAGGATCTGGCATAATCTT TTCCCCCAAGAATAGAAAT T
TT TTCT GCTTAT GAAAGTAGTACATGTT TCTT TAAAAACAAATCAATATT GACT TCTGCCTGCT GTAT
AGCACTATGCCTCCACCTGGCCATGACCAGGGGCATGTCCTGGTCCACCTACCTGAAAATGTTTGCAA

CCAGCCTCCTGGCCATGTGCACAGGGGCTGAAGTTGTCCCACAGGTATTACGGGCCAACCTGACAATA
CATGAAGTTCCACCAAAGTCTGAGAACTCAGAACTGAGCTTTGGGGACTGAAAGACAGCACAAACCTC
AAATTTCTCAGCACTGGAAACCTCAAAATATAACTGAATTCCATAAATAAGATTTTAAGTCTTAAATA
TGTATT TT TAAATGTATTAAAAGTCAAGCTGCTTGTAT TTAAGCACCTAATACAATGCTTAGGT TGTA
AAAGGAGATGCTCAATAGGTACTAACTGATATATTGAGATTTAATTATGGTTTGACCAATATTTATTG
GAAACCGCCAAAGCTTAAATCATCAGCTTCTTGAATGTGATTTGAAAGGTAATTTAGTATTGAATAGC
ATGTGAGCTAGAGTATTTCATTCTTTCTGGTTTATTTCTTCAAATAGACTTTGAATATAATGGTGAAT
GGGT AT TATAAATTAACTAATAAAAATGACAT TGAAAATGAAAAAATATATATATTAAAGTGTAGAAA
GTGACCAGGCGTGGTGGCTCACACCTGTAATCCAAGCACCTTGGGAGGCTGAGGCAGGAGGATCTCTT
GATCCCAGGAGTTCAAGACCAGCCTGGGCAACATAGCGAGACTTCGTCTCT
GAGAG
AGAAAAAAAT TT TT TT TATT TAAAAAAAGTGTAGAAAGTGTCAAGACCCCACTTCT TACCAT TATTTG
GTATAT TTCTCTATACCCACCCACCCTTCCTCCT TACTCCCTCCCTCCCT TCCCAATCTT TT TATCT T
TTTGTATTCTGATTTTTTGTTTGTATATTTTGCTTTAATTTAATGTATCCTTTAAAAATTTCCCATAC
ATTTTATATGTATATATAAAAACGCATGCTGCCAAAGATAATTTATAAGAAAGACCATTGAATTTTTT
TAAAAGT GAT AT AT AT T CAT TGAAAAAAAT TTAGAATATATAGCAAAGCAATAAAGAACTAAATAAAA
TTGCTGTAACTCCTCT TTCAAAGATAAGTGCT TT TATGAT TT TGTTGTAT TT TT TTCTGTATATAGGT
ACATATATAGTATT TATAAAGCTGTACTCATAGTACAT TT TCACATCACAGGTACCATATCAGTGTTA
TTAAATAT TT TGTATGCCAGGGGCTAGACATACCAAGACAACCAATATGTGGTTCTACTTAAATAATA
TTAGAGTATCTTTTATGATGACACTTCATGAGTTGACTATAATAATCTTAGACTTCTAAGAGTTTGGG
TTTTCAAAAGATCACTTAGCTTTTTTGGGTGATTTTTCCCCCTTACTGTGAGATGAGAGAGGCTGTTT
GGAT TTGGGATTGGGGTAGCGGGGACAGCAACTT TI CT TT TCTT TT TCTT TT TTAT TT
TGAGGTAGGG
TATTGCTGTGTCACCCAGGCTGGAGTGCAGTGGTGTGATCTCGGCTCACTGCAACCTCCACCTCCCGG
GCTCAGGTGATCCTCCTGCTTCAGCCTCCCAGTAACTGGGACTACAGGCGCGTGCCACATGCCTGGCT
AATT TTGTAT TT TTAGTAGAGATGGGGT TTCACCATGT TGGCCAGGCTGGTCTCTAACTCCTGACCTC
AGGTGATACGCCCACCTGGGCCTCCCAAAATACTGGGATTACAGGCATGAGCCGCTGCATCAGCCAGC
AGTTTTTCTTGTGGTTTTTTTTGTTTGTTTTGTTTTGTTTTGTTTTTGAGATAGGGTCTTACTCTGTT
GTCCACGCTGGAGTGCTGTGGTATGATCGTAGCTCACTGCAGCCTCAAACTCCTGGGCTCAAGTGATT
CCTTCTGCCTCCGCCTCCCGAGTAGCTGGGACTACAGGTATGCACCACCATACCTGGCAAAT TT TTAC
AAAGTT TT TTGTAGGGACGGGGTCTTGCTACATTCCCCATGTCGGTCT TGAACTCCTGGCCTCAAGCA
ACTCTCCTGTCTCAGCCTCCCAAAGCACTGGGATTACAAGTGTGAGCCACCACACCATGCCAGTTTTT
CCTGTTCAGTGTGATATTTTATCTTGTTAGACTACAGTGTGTTAAAACTTGTTTTACTAAATTTTCAA
ACATACTCAAAAGTGGAGAGAATAGTATAATGAATACCCGTATGTTCATCACCCATGTTTAGAATATT
ATTAAATATAAAGATTTTGCTGCGTTTGTCTTAGCTCTTTAAAATTTTTCTTTTTCTCTTTGTGACCT
AAAGGAAATTCCATATCT TATCACTT TACT TCTACATTCT TGACTAAGATGACTAAGACATATAGTTA
CATGGTTTTTTGTTTTGTTTTTGTTTTTTAAAGACGAAATCTCGCTCTTGTCCCCCAGGCTGGAGTGC
AATGGTGCCATCTCAGCTCAGTGCAACCTCTGCCTTCTGGGTACAAGCGATTCTCCTGCCTCAGCCTC
CCAAGTAGCTGGGATTACAGGCTCCTGCCACCACGCCTGGCTAATTTTTGTATTTTTAGTAGAGACGG
CGGGGGGAGGTTTCACCATGTTGACAAGGCTGGTCTGGAACTCCTGACCTCAGGTGATCCACCCGCCT
CGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCAGCCTGTTTTTTTGTTTGTGTGT
TTTGTTTTTTTTGAGACAGAGTCTTGCTCTGTTTCCCAGGCTGGAGTGAAGTGGTGCCATCTCAGCTC
AGAGACAGAGTCTTGCTCTGTTTCCCAGGCTGGAGTGAAGTGGTGCCATCTTGGCTCACTGCAACCTT
CACCTCCCAGGTTCAAGTGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCATGTGTCA
CCACACCCGGCTAATTTTTTTGTATTTTTAGTAGAGACGGGATTTCACCGTGTTGCCCAGGCTGGTCT

CGAACTCCTGAGCTCAGGCAGICTGCCTGCCTCAGCCTCCCAAAGTGCTGGGATTACACGTGTGAACC
AACCCGCCCGGCCIGTTGITTICTTACATAATTCATTATCATACCTACAAAGTTAACAGTTACTAATA
TCATCT TACACCTAAATT TCTCTGATAGACTAAGGT TATT TT TTAACATCTTAATCCAATCAAATGT T
TGTATCCT GTAATGCTCTCATT GAAACAGCTATATT TCTT TT TCAGAT TAGT GATGAT GAACCAGGT T
ATGACCITGATTTATITTGCATACCTAATCATTATGCTGAGGATTTGGAAAGGGIGITTATTCCTCAT
GGACTAAT TATGGACAGGTAAGTAAGATCT TAAAAT GAGGITTI TTACTT TT TCTT GT GT TAATTTCA
AACATCAGCAGCTGTICTGAGTACTTGCTATTTGAACATAAACTAGGCCAACTTATTAAATAACTGAT
GCTT TCTAAAATCT TCTT TATTAAAAATAAAAGAGGAGGGCCTTACTAAT TACT TAGTATCAGT TGT G
GTATAGIGGGACTCTGTAGGGACCAGAACAAAGTAAACATTGAAGGGAGATGGAAGAAGGAACTCTAG
CCAGAGICTTGCATTICTCAGTCCTAAACAGGGTAATGGACTGGGGCTGAATCACATGAAGGCAAGGT
CAGATT TT TATTAT TATGCACATCTAGCTT GAAAAT TT TCTGTTAAGICAAT TACAGT GAAAAACCT T
ACCT GGTATT GAAT GCTT GCAT TGTATGICTGGCTATTCT GT GT TT TTAT TT TAAAAT
TATAATATCA
AAATAT TT GT GT TATAAAATAT TCTAACTATGGAGGCCATAAACAAGAAGACTAAAGT TCTCTCCTT T
CAGCCTICTGTACACATTICTICTCAAGCACTGGCCTATGCATGTATACTATATGCAAAAGTACATAT
ATACATTTATATITTAACGTATGAGTATAGTITTAAATGTTATTGGACACTITTAATATTAGTGIGTC
TAGAGCTATCTAATATAT TT TAAAGGTT GCATAGCATTCT GTCT TATGGAGATACCATAACT GATTTA
ACCAGTCCACTATT GATAGACACTAT TT TGTICT TACCGACT GTACTAGAAGAAACAT TCTT TTACAT
GT TT GGTACT TGITCAGCTT TATTCAAGTGGAAT TI CT GGGTCAAGGGGAAAGAGT TTAT TGAATAT T

TT GGTATT GCCAAATT TICCICTAAGAAGT TGAATCAT TT TATACTCCTGAT GT TATATGAGAGTACC
TTICTCTICACAATTTGICTCTITTITTITTITTITTGAGACAAGGICTCTGTTGCCCAGGCTGGGGT
GCAGTGCAGCAGAATGATCACAGTICACTGCAGICTCAACCTCCTGGGITCAAGCGATCCTICCACCT
CAGCCTCCTGAGTAGCTGGGACTATAGGTGTGCGCCACCACTCCCAGCTAATATTTTTATTTTGTAGA
AACAGGGT TCGCCATGTTACCCAGCCTCCCAAAGTGCT GGGATTACAGGCAT GAGCCACT GGCCCAGT
TTCTACAGTCTCTCTTAATATTGTATATTATCCAGAAAATTICATTTAATCAGAACCTGCCAGICTGA
IAGGIGAIGGIAICIIGIIIIIAIIIGCAIIIAPAIIAIGAIAGIGGIAIGCIIGGIIII
TTTGAAGGTATCAAATTTTTTACCTTAT GAAACATGAGGGCAAAGGAT GT GATACGTGGAAGATTTAA
AAAAAATT TT TAAT GCAT TT TT TT GAGACAAGGTCT TGCTCTAT TGTCCAGGCT GGAGTGCAGT
GGCA
CAATCACAGT TCACTCCAGCCTCAACATCCTGCACTAAAGTGAT TT TCCCACCTCACCICTCAAGTAG
CIGGGACTACAGGTACATGCTACCATGCCIGGCTAATTITTITTITTITGCAGGCATGGGGICTCACT
ATATTGCCCAGGITGGIGTGGAAGTTTAATGACTAAGAGGIGITTGTTATAAAGTTTAATGTATGAAA
CITTCTATTAAATTCCTGATITTATTICTGTAGGACTGAACGICTTGCTCGAGATGTGATGAAGGAGA
TGGGAGGCCATCACATTGTAGCCCICTGIGTGCTCAAGGGGGGCTATAAATTCTITGCTGACCTGCTG
GATTACATCAAAGCACTGAATAGAAATAGT GATAGATCCATTCCTATGACTGTAGATT TTATCAGACT
GAAGAGCTATTGIGTGAGTATATTTAATATATGATTCTITTTAGTGGCAACAGTAGGITTICTTATAT
TTICITTGAATCTCTGCAAACCATACTTGCTITCATTICACTIGGITACAGTGAGATTITTCTAACAT
AT TCACTAGTACTT TACATCAAAGCCAATACT GT TT TT TTAAAACTAGTCACCT TGGAGGATATATAC
TTATITTACAGGIGTGIGTGGITTITTAAATAAACTCCTITTAGGAATTGCTGTTGGGACTTGGGATA
CT TT TT TCACTATACATACT GGTGACAGATACCCTCTCTT GAGCTACATCGGTT TGIGGGGAGICAAA
AGTCCT TT GGAGCTAGGT TT GACAAATAAGGT GGGT TAACACTT GT TTCCTAGAAAGCACAT GGAGAG
CTAGAGTATT GGCGAATT GAAGAAATCCCCCT TT TT TT TTAACACACT TAAGAAAGGGGACT GCAGGT
ATACTCAAGAGAGTAAGTCGCACCAGAAACCACT TT TGATCCACAGICTGCCIGTGICACACAATTGA
AATGCATCACAACATT GACACT GT GGAT GAAACAAAATCAGT GT GAAT TT TAGTAGTGAATT T CAT T
C
ATAATTTGATCGTGCAAACGITTGATTITTATTACTITAGACTATTGITTCTGATTITATGTTGGGIT

GGTATT TCCTGTGAGT TACTGT TT TACCTT TAAAATAGGAAT TT TTCATACTCT TCAAAGAT TAGAAC
AAATGICCAGTT TT TGCTGT TICATGAATGAGTCCTGICCATCT TIGTAGAAACTCGCCITATGITCA
CATT TT TATTGAGAATAAGACCACTTATCTACAT TTAACTATCAACCTCATCCTCTCCAT TAATCATC
TATT TTAGTGACCCAAGT TT TTGACCTT TTCCATGT TTACATCAATCCTGTAGGTGAT TGGGCAGCCA
TT TAAGTATTAT TATAGACATT TTCACTATCCCATTAAAACCCT TTATGCCCATACATCATAACACTA
CT TCCTACCCATAAGCTCCT TT TAACTTGT TAAAGTCT TGCT TGAATTAAAGACTTGT TTACGGTATC
GATAAGCT T GAT AT CAAAAC GC CAAC T T T GAC CC GGAACGCGGAAAACAC CT
GAGAAAAACACCTGGG
CGAGTCTCCACGTAAACGGTCAAAGTCCCCGCGGCCCTAGACAAATATTACGCGCTATGAGTAACACA
AAAT TATTCAGATT TCAC TTCC TC TTAT TCAG TT TTCC CGCGAAAATGGC CAAATC TTAC TC GG
TTAC
GC CCAAAT TTAC TACAACATCC GC C TAAAACC GC GC GAAAAT TG TCAC TTCC TG TG TACAC C
GG C G CA
CACCAAAAACGTCACT TT TGCCACATCCGTCGCT TACATGTGTTCCGCCACACT TGCAACATCACACT
TCCGCCACACTACTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTC
AT TATCATAT TGGC TTCAATCCAAAATAAGGTATAT TATTGATGATGT T T AAACAT TAAGAATTAATT
CGATCCTGAATGGCGAATGGACGCGCCCTGTAGCGGCGCATTAAGCGCGCGGGTGTGGTGGTTACGCG
CAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCG
CCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGAGCT
TTACGGCACCTCGACCGCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATA
GACGGT TT TTCGCCCT TTGACGTTGGAGTCCACGTTCT TTAATAGTGGACTCTTGT TCCAAACTGGAA
CAACACTCAACCCTATCGCGGTCTATTCTTTTGATTTATAAGGGATGTTGCCGATTTCGGCCTATTGG
TTAAAAAATGAGCTGATT TAACAAAAAT TT TAACAAAATTCAGAAGAACTCGTCAAGAAGGCGATAGA
AGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCG
CCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCG
GCCACAGTCGATGAATCCAGAAAAGCGGCCAT TT TCCACCATGATATTCGGCAAGCAGGCATCGCCAT
GGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCG
AGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCG
CTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCA
TTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGC
ACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAAC
GCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGT
CGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCG
ATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAA
TCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGAGCTTGATCCCCTGCGCC
ATCAGATCCT TGGCGGCAAGAAAGCCATCCAGTT TACT TTGCAGGGCT TCCCAACCTTACCAGAGGGC
GCCCCAGCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCCCAGTCTAGCTATCGCCATGTAAG
CCCACTGCAAGCTACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGA
CATTCATCCGGGGICAGCACCGTTICTGCGGACTGGCTTICTACGTGAAAAGGATCTAGGTGAAGATC
CT TT TTGATAATCTCATGGCTGCAGCAATGGCAACAACGT TGCGCAAACTAT TAACTGGCGAACTACT
TACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGC
GCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGT
ATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCA
GGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAAC
TGTCAGACCAAGTT TACT CATATATACT TTAGAT TGAT TTAAAACT TCAT TT TTAATT TAAAAGGATC

TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGC
GTCAGAC
RightITR = first underlined and bold sequence CBh = first underlined sequence mCherry:PKD1 = first bold sequence HGHpA = second underlined sequence Packaging Signal = second bold sequence LeftITR = second underlined and bold sequence SEQ ID NO:6 LeftITR-EF 1 a-PKD 2-B GHpA-RightITR
AGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCA
CCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTT
CAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACT
CTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG
TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGG
GGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC
TATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGA
ACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG
CCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCA
GCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTCCTGCAGGCAGCTG
CGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGC
CTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCA
CGCGTTAACTATAACGGTCCTAAGGTAGCGAAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCG
CCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGG
GGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATA
TAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCC
GTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCAC
CTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTT
GCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTG
CGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTG
ATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTG
GTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGG
CGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGT
GCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTG
CGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGAAGGACGCGGCGCTCG
GGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATG

TGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGT
CT TTAGGT TGGGGGGAGGGGTT TTATGCGATGGAGT TTCCCCACACTGAGTGGGTGGAGACTGAAGT T
AGGCCAGCTTGGCACT TGATGTAATTCTCCTIGGAATT TGCCCT TT TTGAGT TTGGATCT TGGT TCAT
TCTCAAGCCTCAGACAGIGGITCAAAGT TT TT TI CT TCCATT TCAGGIGTCGTGATCCGGAGGCGGCG
GCACGGGCGGCGGCAGCGGCGGCATGGTGAACTCCAGTCGCGTGCAGCCTCAGCAGCCCGGGGACGCC
AAGCGGCCGCCCGCGCCCCGCGCGCCGGACCCGGGCCGGCTGATGGCTGGCTGCGCGGCCGTGGGCGC
CAGCCTCGCCGCCCCGGGCGGCCTCTGCGAGCAGCGGGGCCTGGAGATCGAGATGCAGCGCATCCGGC
AGGCGGCCGCGCGGGACCCCCCGGCCGGAGCCGCGGCCTCCCCTTCTCCTCCGCTCTCGTCGTGCTCC
CGGCAGGC GTGGAGCC GC GATAAC CC CGGC TTCGAGGC CGAGGAGGAGGAGGAGGAGG TGGAAGGGGA
AGAAGGCGGAATGGTGGTGGAGATGGACGTAGAGTGGCGCCCGGGCAGCCGGAGGTCGGCCGCCTCCT
CGGCCGTGAGCTCCGTGGGCGCGCGGAGCCGGGGGCTTGGGGGCTACCACGGCGCGGGCCACCCGAGC
GGGAGGCGGCGCCGGCGAGAGGACCAGGGCCCGCCGTGCCCCAGCCCAGTCGGCGGCGGGGACCCGCT
GCATCGCCACCTCCCCCTGGAAGGGCAGCCGCCCCGAGTGGCCTGGGCGGAGAGGCTGGTTCGCGGGC
TGCGAGGTCTCTGGGGAACAAGACTCATGGAGGAAAGCAGCACTAACCGAGAGAAATACCTTAAAAGT
GT TT TACGGGAAC TGG TCACATAC C TCC TT TT TC TCATAG TC TTGTGCATC T TGAC C TAC
GGCATGAT
GAGC TC CAATGTGTAC TAC TACAC CC GGATGATG TCACAGC TC T TC C TAGACAC CC CC
GTGTCCAAAA
CGGAGAAAAC TAAC TT TAAAAC TC TG TC TTCCATGGAAGAC T TC TGGAAG TTCACAGAAGGC TC C
TTA
TTGGATGGGCTGTACTGGAAGATGCAGCCCAGCAACCAGACTGAAGCTGACAACCGAAGTTTCATCTT
C TATGAGAAC C TGC TG TTAGGGGT TC CACGAATACGGCAAC TCC GAGTCAGAAATGGATC C TGC TC
TA
TC CC CCAGGAC T TGAGAGATGAAATTAAAGAG TGC TATGATG TC TAC TC TGTCAGTAG
TGAAGATAGG
GC TC CC TT TGGGCC CC GAAATGGAAC CGC T TGGATC TACACAAG TGAAAAAGAC TTGAATGG
TAGTAG
CCAC TGGGGAATCATTGCAAC T TATAGTGGAGC TGGC TAT TATC TGGATT TG TCAAGAACAAGAGAGG
AAACAGCTGCACAAGTTGCTAGCCTCAAGAAAAATGTCTGGCTGGACCGAGGAACCAGGGCAACTTTT
AT TGAC TTCTCAGTGTACAACGCCAACATTAACC TGTTCTGTGTGGTCAGGT TATTGGTTGAAT TCCC
AGCAACAGGTGGTGTGATTCCATCTTGGCAATTTCAGCCTTTAAAGCTGATCCGATATGTCACAACTT
TTGATT TC TTCC TGGCAGCC TGTGAGAT TATC TT TTGT TTCT TTATCT TT
TACTATGTGGTGGAAGAG
ATATTGGAAATTCGCATTCACAAACTACACTATTTCAGGAGTTTCTGGAATTGTCTGGATGTTGTGAT
CGTTGTGCTGTCAGTGGTAGCTATAGGAATTAACATATACAGAACATCAAATGTGGAGGTGCTACTAC
AG TT TC TGGAAGATCAAAATAC TT TC CC CAAC TT TGAGCATC TGGCATAT TGGCAGATACAG
TTCAAC
AATATAGC TGC TGTCACAGTAT TT TT TG TC TGGATTAAGC TC TTCAAATTCATCAATT TTAACAGGAC

CATGAGCCAGCTCTCGACAACCATGTCTCGATGTGCCAAAGACC TGTT TGGC TT TGCTAT TATGTTC T
TCATTATTTTCCTAGCGTATGCTCAGTTGGCATACCTTGTCTTTGGCACTCAGGTCGATGACTTCAGT
AC TT TC CAAGAG TG TATC TTCAC TCAAT TC CG TATCAT TT TGGGCGATATCAAC TT
TGCAGAGATTGA
GGAAGCTAATCGAGTTTTGGGACCAATTTATTTCACTACATTTGTGTTCTTTATGTTCTTCATTCTTT
TGAATATG TT TT TGGC TATCATCAATGATAC T TAC TC TGAAG TGAAATC TGAC T
TGGCACAGCAGAAA
GC TGAAATGGAAC TC TCAGATC TTATCAGAAAGGGC TACCATAAAGC T TTGG TCAAAC TAAAAC TGAA

AAAAAATACC GTGGATGACATT TCAGAGAG TC TGCGGCAAGGAGGAGGCAAG TTAAAC TT TGAC GAAC
TTCGACAAGATCTCAAAGGGAAGGGCCATACTGATGCAGAGATTGAGGCAATATTCACAAAGTACGAC
CAAGATGGAGACCAAGAACTGACCGAACATGAACATCAGCAGATGAGAGACGACTTGGAGAAAGAGAG
GGAGGACC TGGATT TGGATCACAG TTC T TTAC CACG TC CCATGAGCAGCC GAAG TT TC CC TC
GAAGC C
TGGATGACTCTGAGGAGGATGACGATGAAGATAGCGGACATAGCTCCAGAAGGAGGGGAAGCATTTCT
AG TGGC GT TTC T TACGAAGAGT TTCAAG TC C TGG TGAGAC GAGTGGAC CGGATGGAGCAT TC
CATCGG
CAGCATAG TG TC CAAGAT TGAC GC CG TGATCG TGAAGC TAGAGATTATGGAGCGAGCCAAAC TGAAGA

GGAGGGAGGTGCTGGGAAGGCTGTTGGATGGGGTGGCCGAGGATGAAAGGCTGGGTCGTGACAGTGAA
ATCCATAGGGAACAGATGGAACGGCTAGTACGTGAAGAGTTGGAACGCTGGGAATCCGATGATGCAGC
TTCCCAGATCAGTCATGGTTTAGGCACGCCAGTGGGACTAAATGGTCAACCTCGCCCCAGAAGCTCCC
GCCCATCTTCCTCCCAATCTACAGAAGGCATGGAAGGTGCAGGTGGAAATGGGAGTTCTAATGTCCAC
GTATGATTCTAGAGTCGACCTGCAGAAGCTTGCCTCGAGCCTGTGCCTTCTAGTTGCCAGCCATCTGT
TGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAA
ATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGAC
AGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGTAACTAT
AACGGTCCTAAGGTAGCGAACGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCACTCCC
TCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCG
GGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCTGCCTGCAGGGGCGCCTGATGCGGTATTTTCTCCTTA
CGCATCTGTGCGGTATTTCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCAT
TAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCT
CCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGG
GCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTTGGGTGATG
GTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTT
AATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGATTTATA
AGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATT
TTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAG
TTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATC
CGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGA
AACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTT
TCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAAT
ACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGA
AGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTT
TTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTA
CATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGA
TGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTC
GGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTAC
GGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACT
TACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTA
ACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGAT
GCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGC
AACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCT
GGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGG
GCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAAC
GAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTAC
TCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTT
TGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAA
RightITR = first underlined and bold sequence EFla = first underlined sequence PKD2 = bold sequence BGHpA = second underlined sequence LeftITR = second underlined and bold sequence SEQ ID NO: 7 HDAd-PKD1-PKD2 RightITR-CBh-mCherry:PKD1-HGHpA-EFla-PKD2-BGHpA-PackagingSignal-LeftITR
CCCGTAGAAAAGATCAAAGGATCTICTIGAGATCCITITITICTGCGCGTAATCTGCTGCTIGCAAAC
AAAAAAACCACCGCTACCAGCGGIGGITTGITTGCCGGATCAAGAGCTACCAACTCTITTICCGAAGG
TAACTGGCTICAGCAGAGCGCAGATACCAAATACTGICCTICTAGIGTAGCCGTAGITAGGCCACCAC
TICAAGAACTCTGTAGCACCGCCIACATACCICGCTCTGCTAATCCIGITACCAGTGGCTGCTGCCAG
TGGCGATAAGICGTGICITACCGGGITGGACTCAAGACGATAGITACCGGATAAGGCGCAGCGGICGG
GCTGAACGGGGGGITCGTGCACACAGCCCAGCTIGGAGCGAACGACCIACACCGAACTGAGATACCIA
CAGCGTGAGCTATGAGAAAGCGCCACGCTICCCGAAGGGAGAAAGGCGGACAGGIATCCGGTAAGCGG
CAGGGICGGAACAGGAGAGCGCACGAGGGAGCTICCAGGGGGAAACGCCIGGIATCTITATAGICCIG
TCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGG
AAAAACGCCAGCAACGCGGCCITITTACGGITCCIGGCCITTIGCTGGCCITTIGCTCACATGITCTT
TCCIGCGTTATCCCCTGATTCTGIGGATAACCGTATTACCGCCITTGAGTGAGCTGATACCGCTCGCC
GCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTT
CICCITACGCATCTGIGCGGIATTICACACCGCATATGGATCCATGCATGITAAGITTAAACATCATC
AATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAGTTTGTGACGTGGCGCG
GGGCGTGGGAACGGGGCGGGTGACGTAGGTTTTAGGGCGGAGTAACTTGTATGTGTTGGGAATTGTAG
TITICTTAAAATGGGAAGITACGTAACGIGGGAAAACGGAAGTGACGATTTGAGGAAGITGIGGGITT
TTIGGCTITCGTTICTGGGCGTAGGITCGCGTGCGGITTICTGGGIGITITTIGIGGACTITAACCGT
TACGTCATTTTTTAGTCCTATATATACTCGCTCTGCACTTGGCCCTTTTTTACACTGTGACTGATTGA
GCTGGIGCCGTGICGAGIGGIGITTITTGATGCCCCCCCICGAGGITCGACGGIATCGATAAGCTIGA
TTTAATTAAGGCCGGCCCCTAGGGGCGCGCGCGGCCGCTAGGGATAACAGGGTAATTGTTGACAATTA
ATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGIGAGGAACTAAACCATGGCCAAGITGA
CCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTC
GGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCAT
CAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACG
AGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACC
GAGATCGGCGAGCAGCCGIGGGGGCGGGAGITCGCCCIGCGCGACCCGGCCGGCAACTGCGTGCACTT
CGIGGCCGAGGAGCAGGACTGAACGCGICGTTACATAACTTACGGTAAATGGCCCGCCIGGCTGACCG
CCCAACGACCCCCGCCCATTGACGICAATAATGACGTATGITCCCATAGTAACGCCAATAGGGACTIT
CCATTGACGICAATGGGIGGAGTATTTACGGTAAACTGCCCACTIGGCAGTACATCAAGIGTATCATA
TGCCAAGTACGCCCCCIATTGACGICAATGACGGTAAATGGCCCGCCIGGCATTATGCCCAGTACATG
ACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGT
GAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTA
ITTITTAATTATITTGIGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGG

CGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCG
AAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGG
GAGTCGCTGCGACGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCT
CTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGC
TGAGCAAGAGGTAAGGGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCAC
CTGICCGGAGAATT CGCCACCATGCCGCCCGCCGCGCCCGCCCGCCTGGCGCTGGCCCTGGGCCTGGG
_ CC TG TGGC TC GGGGCGC TGGCGGGGGGC CC CGGGATGG TGAGCAAGGGCGAGGAGGATAACATGGCCA
TCATCAAGGAGT TCATGC GC TTCAAGGTGCACATGGAGGGC TCC GTGAAC GGCCAC GAGT TC GAGATC
GAGGGC GAGGGC GAGGGC CGCC CC TACGAGGGCACC CAGACC GC CAAGC TGAAGGTGACCAAGGGTGG
CCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGC
ACCCCGCCGACATCCCCGAC TACT TGAAGC TGTCCT TCCCCGAGGGCT TCAAGTGGGAGCGCGTGATG
AAC T TC GAGGAC GGCGGC GTGG TGAC CG TGAC CCAGGAC TCC TC CC TGCAGGAC GGCGAG
TTCATC TA
CAAGGTGAAGC TGC GC GGCACCAAC T TC CC C TCC GACGGC CC CG TAATGCAGAAGAAGAC
CATGGGC T
GGGAGGCC TC C TCC GAGC GGATGTAC CC CGAGGACGGC GC CC TGAAGGGC GAGATCAAGCAGAGGC
TG
AAGC TGAAGGAC GGCGGC CAC TAC GACGC TGAGG TCAAGACCAC C TACAAGGCCAAGAAGCC CG
TGCA
GC TGCC CGGC GC C TACAACG TCAACATCAAGT TGGACATCAC C TCC CACAAC GAGGAC
TACACCATC G
TGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGGGCGCG
CCGGGGGGCCCCGGGCGCGGCTGCGGGCCCTGCGAGCCCCCCTGCCTCTGCGGCCCAGCGCCCGGCGC
CGCCTGCCGCGTCAACTGCTCGGGCCGCGGGCTGCGGACGCTCGGTCCCGCGCTGCGCATCCCCGCGG
ACGCCACAGCGCTAGACGTCTCCCACAACCTGCTCCGGGCGCTGGACGTTGGGCTCCTGGCGAACCTC
TC GGCGC TGGCAGAGC TGGATATAAGCAACAACAAGAT TTC TAC GT TAGAAGAAGGAATATT TGC TAA
TT TATT TAAT TTAAGTGAAATAAACC TGAG TGGGAACC CG TT TGAG TG TGAC TG TGGC C TGGCG
TGGC
TGCC GC GATGGGCGGAGGAGCAGCAGGTGC GGGTGG TGCAGC CC GAGGCAGC CACG TG TGC TGGGCC
T
GGCTCCCTGGCTGGCCAGCCTCTGCTTGGCATCCCCTTGCTGGACAGTGGCTGTGGTGAGGAGTATGT
CGCC TGCC TCCC TGACAACAGC TCAGGCACCGTGGCAGCAGTGTCC TT TTCAGC TGCCCACGAAGGCC
TGCTTCAGCCAGAGGCCTGCAGCGCCTTCTGCTTCTCCACCGGCCAGGGCCTCGCAGCCCTCTCGGAG
CAGGGCTGGTGCCTGTGTGGGGCGGCCCAGCCCTCCAGTGCCTCCTTTGCCTGCCTGTCCCTCTGCTC
CGGCCCCCCGCCACCTCCTGCCCCCACCTGTAGGGGCCCCACCCTCCTCCAGCACGTCTTCCCTGCCT
CCCCAGGGGCCACCCTGGTGGGGCCCCACGGACCTCTGGCCTCTGGCCAGCTAGCAGCCTTCCACATC
GC TGCCCCGC TCCC TGTCAC TGCCACACGC TGGGAC TTCGGAGACGGC TCCGCCGAGGTGGATGCCGC
TGGGCCGGCTGCCTCGCATCGCTATGTGCTGCCTGGGCGCTATCACGTGACGGCCGTGCTGGCCCTGG
GGGCCGGCTCAGCCCTGCTGGGGACAGACGTGCAGGTGGAAGCGGCACCTGCCGCCCTGGAGCTCGTG
TGCC CG TC C TCGGTGCAGAG TGAC GAGAGC C T TGAC C TCAGCATCCAGAACC GC GG TGGT
TCAGGCC T
GGAGGCCGCCTACAGCATCGTGGCCCTGGGCGAGGAGCCGGCCCGAGCGGTGCACCCGCTCTGCCCCT
CGGACACGGAGATCTTCCCTGGCAACGGGCACTGCTACCGCCTGGTGGTGGAGAAGGCGGCCTGGCTG
CAGGCGCAGGAGCAGTGTCAGGCC TGGGCC GGGGCC GC CC TGGCAATGGTGGACAG TC CC GC CG TGCA

GCGCTTCCTGGTCTCCCGGGTCACCAGGAGCCTAGACGTGTGGATCGGCTTCTCGACTGTGCAGGGGG
TGGAGG TGGGCC CAGC GC CGCAGGGC GAGGCC TTCAGC C TGGAGAGC TGC CAGAAC TGGC TGCC
CGGG
GAGCCACACCCAGCCACAGCCGAGCACTGCGTCCGGCTCGGGCCCACCGGGTGGTGTAACACCGACCT
GTGC TCAGCGCC GCACAGC TAC GTC TGC GAGC TGCAGC CC GGAGGC CCAG TGCAGGATGC
CGAGAAC C
TCCTCGTGGGAGCGCCCAGTGGGGACCTGCAGGGACCCCTGACGCCTCTGGCACAGCAGGACGGCCTC
TCAGCCCCGCACGAGCCCGTGGAGGTCATGGTATTCCCGGGCCTGCGTCTGAGCCGTGAAGCCTTCCT
CACCACGGCCGAATTTGGGACCCAGGAGCTCCGGCGGCCCGCCCAGCTGCGGCTGCAGGTGTACCGGC

TC C TCAGCACAGCAGGGACC CC GGAGAACGGCAGCGAGCC TGAGAGCAGG TC CC CGGACAACAGGAC C
CAGCTGGCCCCCGCGTGCATGCCAGGGGGACGCTGGTGCCCTGGAGCCAACATCTGCTTGCCGCTGGA
CGCCTCCTGCCACCCCCAGGCCTGCGCCAATGGCTGCACGTCAGGGCCAGGGCTACCCGGGGCCCCCT
ATGCGCTATGGAGAGAGTTCCTCTTCTCCGTTCCCGCGGGGCCCCCCGCGCAGTACTCGGTCACCCTC
CACGGCCAGGATGTCCTCATGCTCCCTGGTGACCTCGTTGGCTTGCAGCACGACGCTGGCCCTGGCGC
CC TCCTGCAC TGCTCGCCGGCTCCCGGCCACCCTGGTCCCCAGGCCCCGTACCTCTCCGCCAACGCC T
CGTCATGGCTGCCCCACTTGCCAGCCCAGCTGGAGGGCACTTGGGCCTGCCCTGCCTGTGCCCTGCGG
CTGCTTGCAGCCACGGAACAGCTCACCGTGCTGCTGGGCTTGAGGCCCAACCCTGGACTGCGGCTGCC
TGGGCGCTATGAGGTCCGGGCAGAGGTGGGCAATGGCGTGTCCAGGCACAACCTCTCCTGCAGCTTTG
ACGTGGTCTCCCCAGTGGCTGGGCTGCGGGTCATCTACCCTGCCCCCCGCGACGGCCGCCTCTACGTG
CCCACCAACGGCTCAGCCTTGGTGCTCCAGGTGGACTCTGGTGCCAACGCCACGGCCACGGCTCGCTG
GCCTGGGGGCAGTGTCAGCGCCCGCTTTGAGAATGTCTGCCCTGCCCTGGTGGCCACCTTCGTGCCCG
GC TGCC CC TGGGAGAC CAAC GATACC C TGT TC TCAG TGGTAGCAC TGC CG TGGC TCAG
TGAGGGGGAG
CACG TGGTGGAC GTGG TGGTGGAAAACAGC GC CAGC CGGGCCAACC TCAGCC TGCGGG TGAC GGCGGA
GGAGCCCATCTGTGGCCTCCGCGCCACGCCCAGCCCCGAGGCCCGTGTACTGCAGGGAGTCCTAGTGA
GG TACAGC CC CG TGGTGGAGGC CGGC TC GGACATGG TC TTCC GG TGGACCATCAAC
GACAAGCAGTC C
C TGACC TTCCAGAACG TGGTC T TCAATG TCAT TTATCAGAGC GC GGCGGTC T TCAAGC TC TCAC
TGAC
GGCCTCCAACCACGTGAGCAACGTCACCGTGAACTACAACGTAACCGTGGAGCGGATGAACAGGATGC
AGGGTCTGCAGGTCTCCACAGTGCCGGCCGTGCTGTCCCCCAATGCCACGCTAGCACTGACGGCGGGC
GTGCTGGTGGACTCGGCCGTGGAGGTGGCCTTCCTGTGGACCTTTGGGGATGGGGAGCAGGCCCTCCA
CCAGTTCCAGCCTCCGTACAACGAGTCCTTCCCGGTTCCAGACCCCTCGGTGGCCCAGGTGCTGGTGG
AGCACAATGTCATGCACACC TACGC TGC CC CAGG TGAG TACC TC C TGACC GTGC TGGCATC
TAATGC C
TTCGAGAACCTGACGCAGCAGGTGCCTGTGAGCGTGCGCGCCTCCCTGCCCTCCGTGGCTGTGGGTGT
GAGTGACGGCGTCCTGGTGGCCGGCCGGCCCGTCACCTTCTACCCGCACCCGCTGCCCTCGCCTGGGG
GTGTTCTTTACACGTGGGACTTCGGGGACGGCTCCCCTGTCCTGACCCAGAGCCAGCCGGCTGCCAAC
CACACC TATGCC TC GAGGGGCACC TACCAC GTGC GC C TGGAGGTCAACAACACGGTGAGC GG TGCGGC

GGCCCAGGCGGATGTGCGCGTCTTTGAGGAGCTCCGCGGACTCAGCGTGGACATGAGCCTGGCCGTGG
AGCAGGGCGCCCCCGTGGTGGTCAGCGCCGCGGTGCAGACGGGCGACAACATCACGTGGACCTTCGAC
ATGGGGGACGGCACCGTGCTGTCGGGCCCGGAGGCAACAGTGGAGCATGTGTACCTGCGGGCACAGAA
CTGCACAGTGACCGTGGGTGCGGCCAGCCCCGCCGGCCACCTGGCCCGGAGCCTGCACGTGCTGGTCT
TCGTCCTGGAGGTGCTGCGCGTTGAACCCGCCGCCTGCATCCCCACGCAGCCTGACGCGCGGCTCACG
GCCTACGTCACCGGGAACCCGGCCCACTACCTCTTCGACTGGACCTTCGGGGATGGCTCCTCCAACAC
GACCGTGCGGGGGTGCCCGACGGTGACACACAACTTCACGCGGAGCGGCACGTTCCCCCTGGCGCTGG
TGC TGTCCAGCC GC GTGAACAGGGCGCATTAC TTCACCAGCATC TGCG TGGAGC CAGAGG TGGGCAAC
GTCACC C TGCAGCCAGAGAGGCAG TT TG TGCAGC TC GGGGAC GAGGCC TGGC TGGTGGCATG TGCC
TG
GCCCCCGTTCCCCTACCGCTACACCTGGGACTTTGGCACCGAGGAAGCCGCCCCCACCCGTGCCAGGG
GC CC TGAGGTGACG TTCATC TACC GAGACC CAGGC TCC TATC TTGTGACAGTCACC GC
GTCCAACAAC
ATCTCTGCTGCCAATGACTCAGCCCTGGTGGAGGTGCAGGAGCCCGTGCTGGTCACCAGCATCAAGGT
CAATGGCTCCCTTGGGCTGGAGCTGCAGCAGCCGTACCTGTTCTCTGCTGTGGGCCGTGGGCGCCCCG
CCAGCTACCTGTGGGATCTGGGGGACGGTGGGTGGCTCGAGGGTCCGGAGGTCACCCACGCTTACAAC
AGCACAGG TGAC TTCACC GT TAGGGTGGCC GGC TGGAATGAGGTGAGC CGCAGC GAGGCC TGGC TCAA

TGTGACGGTGAAGCGGCGCGTGCGGGGGCTCGTCGTCAATGCAAGCCGCACGGTGGTGCCCCTGAATG
GGAGCG TGAGC T TCAGCACG TC GC TGGAGGCC GGCAGTGATG TGCGC TAT TC C TGGGTGC TC TG
TGAC

CGCTGCACGCCCATCCCTGGGGGTCCTACCATCTCTTACACCTTCCGCTCCGTGGGCACCTTCAATAT
CATCGTCACGGCTGAGAACGAGGTGGGCTCCGCCCAGGACAGCATCTTCGTCTATGTCCTGCAGCTCA
TAGAGGGGC TGCAGGTGG TGGGCGGTGGCC GC TAC T TC CC CACCAACCACAC GG TACAGC TGCAGGC
C
GTGG TTAGGGATGGCACCAACG TC TC C TACAGC TGGAC TGCC TGGAGGGACAGGGGCC CGGC CC
TGGC
CGGCAGCGGCAAAGGCTTCTCGCTCACCGTGCTCGAGGCCGGCACCTACCATGTGCAGCTGCGGGCCA
CCAACATGCTGGGCAGCGCCTGGGCCGACTGCACCATGGACTTCGTGGAGCCTGTGGGGTGGCTGATG
GTGGCCGCCTCCCCGAACCCAGCTGCCGTCAACACAAGCGTCACCCTCAGTGCCGAGCTGGCTGGTGG
CAGTGGTGTCGTATACACTTGGTCCTTGGAGGAGGGGCTGAGCTGGGAGACCTCCGAGCCATTTACCA
CCCATAGCTTCCCCACACCCGGCCTGCACTTGGTCACCATGACGGCAGGGAACCCGCTGGGCTCAGCC
AACGCCACCGTGGAAGTGGATGTGCAGGTGCCTGTGAGTGGCCTCAGCATCAGGGCCAGCGAGCCCGG
AGGCAGCT TCGTGGCGGCCGGGTCCTCTGTGCCC TT TTGGGGGCAGCTGGCCACGGGCACCAATGTGA
GC TGGTGC TGGGCTGTGCCCGGCGGCAGCAGCAAGCGTGGCCCTCATGTCACCATGGTCT TCCCGGAT
GC TGGCAC C T TC TC CATC CGGC TCAATGCC TC CAAC GCAG TCAGC TGGGTC TCAGC CACG
TACAACC T
CACGGCGGAGGAGCCCATCGTGGGCCTGGTGCTGTGGGCCAGCAGCAAGGTGGTGGCGCCCGGGCAGC
TGGTCCAT TT TCAGATCC TGCTGGCTGCCGGC TCAGCTGTCACC TTCCGCCTGCAGGTCGGCGGGGCC
AACCCCGAGGTGCTCCCCGGGCCCCGTTTCTCCCACAGCTTCCCCCGCGTCGGAGACCACGTGGTGAG
CGTGCGGGGCAAAAACCACGTGAGCTGGGCCCAGGCGCAGGTGCGCATCGTGGTGCTGGAGGCCGTGA
GTGGGC TGCAGG TGCC CAAC TGC TGC GAGC C TGGCATC GC CACGGGCAC TGAGAGGAAC T
TCACAGC C
CGCGTGCAGCGCGGCTCTCGGGTCGCCTACGCCTGGTACTTCTCGCTGCAGAAGGTCCAGGGCGACTC
GC TGGTCATCCTGTCGGGCCGCGACGTCACCTACACGCCCGTGGCCGCGGGGCTGT TGGAGATCCAGG
TGCGCGCCTTCAACGCCCTGGGCAGTGAGAACCGCACGCTGGTGCTGGAGGTTCAGGACGCCGTCCAG
TATGTGGCCCTGCAGAGCGGCCCCTGCTTCACCAACCGCTCGGCGCAGTTTGAGGCCGCCACCAGCCC
CAGC CC CC GGCG TG TGGC C TAC CAC TGGGAC T
TTGGGGATGGGTCGCCAGGGCAGGACACAGATGAGC
CCAGGGCCGAGCACTCCTACCTGAGGCCTGGGGACTACCGCGTGCAGGTGAACGCCTCCAACCTGGTG
AGCTTCTTCGTGGCGCAGGCCACGGTGACCGTCCAGGTGCTGGCCTGCCGGGAGCCGGAGGTGGACGT
GG TC C TGC CC C TGCAGGTGC TGATGC GGCGATCACAGC GCAAC TAC TTGGAGGC CCAC GT TGAC
C TGC
GC GAC TGC GTCACC TACCAGAC TGAG TACC GC TGGGAGGTGTATCGCACC GC CAGC TGCCAGCGGCC
G
GGGCGCCCAGCGCGTGTGGCCCTGCCCGGCGTGGACGTGAGCCGGCCTCGGCTGGTGCTGCCGCGGCT
GGCGCTGCCTGTGGGGCACTACTGCTTTGTGTTTGTCGTGTCATTTGGGGACACGCCACTGACACAGA
GCATCCAGGCCAATGTGACGGTGGCCCCCGAGCGCCTGGTGCCCATCATTGAGGGTGGCTCATACCGC
GTGTGGTCAGACACACGGGACCTGGTGCTGGATGGGAGCGAGTCCTACGACCCCAACCTGGAGGACGG
CGACCAGACGCCGCTCAGTTTCCACTGGGCCTGTGTGGCTTCGACACAGAGGGAGGCTGGCGGGTGTG
CGCTGAAC TT TGGGCCCCGCGGGAGCAGCACGGTCACCAT TCCACGGGAGCGGC TGGCGGCTGGCGTG
GAGTACACCTTCAGCCTGACCGTGTGGAAGGCCGGCCGCAAGGAGGAGGCCACCAACCAGACGGTGCT
GATCCGGAGTGGCCGGGTGCCCATTGTGTCCTTGGAGTGTGTGTCCTGCAAGGCACAGGCCGTGTACG
AAGTGAGC CGCAGC TC C TAC GTGTAC TTGGAGGGCC GC TGCC TCAATTGCAGCAGC GGC
TCCAAGCGA
GGGC GG TGGGC TGCAC GTAC GT TCAGCAACAAGACGC TGG TGC TGGATGAGACCAC CACATC
CACGGG
CAGTGCAGGCATGCGACTGGTGCTGCGGCGGGGCGTGCTGCGGGACGGCGAGGGATACACCTTCACGC
TCACGGTGCTGGGCCGCTCTGGCGAGGAGGAGGGCTGCGCCTCCATCCGCCTGTCCCCCAACCGCCCG
CCGCTGGGGGGCTCTTGCCGCCTCTTCCCACTGGGCGCTGTGCACGCCCTCACCACCAAGGTGCACTT
CGAATGCACGGGCTGGCATGACGCGGAGGATGCTGGCGCCCCGCTGGTGTACGCCCTGCTGCTGCGGC
GC TG TC GC CAGGGC CAC TGC GAGGAG TTC TGTGTC TACAAGGGCAGCC TC TC CAGC TACGGAGC
CGTG
CTGCCCCCGGGTTTCAGGCCACACTTCGAGGTGGGCCTGGCCGTGGTGGTGCAGGACCAGCTGGGAGC

CGC TGTGG TC GC CC TCAACAGG TC TT TGGC CATCAC CC TC CCAGAGCC CAAC GGCAGC
GCAACGGGGC
TCACAGTCTGGCTGCACGGGCTCACCGCTAGTGTGCTCCCAGGGCTGCTGCGGCAGGCCGATCCCCAG
CACGTCATCGAGTACTCGTTGGCCCTGGTCACCGTGCTGAACGAGTACGAGCGGGCCCTGGACGTGGC
GGCAGAGCCCAAGCACGAGCGGCAGCACCGAGCCCAGATACGCAAGAACATCACGGAGACTCTGGTGT
CC C TGAGGGTCCACAC TG TGGATGACATCCAGCAGATC GC TGC TGC GC TGGC CCAG TGCATGGGGCC
C
AGCAGGGAGC TC GTATGC CGC TCG TGCC TGAAGCAGAC GC TGCACAAGC TGGAGGC CATGATGC
TCAT
CC TGCAGGCAGAGACCAC CGCGGGCACC GTGACGCC CACC GC CATC GGAGACAGCATC C TCAACATCA
CAGGAGAC C TCATC CACC TGGC CAGC TC GGAC GTGC GGGCAC CACAGC CC TCAGAGC TGGGAGC
CGAG
TCACCATCTCGGATGGTGGCGTCCCAGGCCTACAACCTGACCTCTGCCCTCATGCGCATCCTCATGCG
CTCCCGCGTGCTCAACGAGGAGCCCCTGACGCTGGCGGGCGAGGAGATCGTGGCCCAGGGCAAGCGCT
CGGACCCGCGGAGCCTGCTGTGCTATGGCGGCGCCCCAGGGCCTGGCTGCCACTTCTCCATCCCCGAG
GC TT TCAGCGGGGCCC TGGCCAACCTCAGTGACGTGGTGCAGCTCATC TT TC TGGTGGAC TCCAATCC
CTTTCCCTTTGGCTATATCAGCAACTACACCGTCTCCACCAAGGTGGCCTCGATGGCATTCCAGACAC
AGGC CGGC GC CCAGATCC CCATCGAGCGGC TGGC C TCAGAGC GC GC CATCAC CG TGAAGG TGCC
CAAC
AACTCGGACTGGGCTGCCCGGGGCCACCGCAGCTCCGCCAACTCCGCCAACTCCGTTGTGGTCCAGCC
CCAGGCCTCCGTCGGTGCTGTGGTCACCCTGGACAGCAGCAACCCTGCGGCCGGGCTGCATCTGCAGC
TCAAC TATAC GC TGC TGGAC GGCCAC TACC TG TC TGAGGAAC C TGAGC CC TACC TGGCAG TC
TACC TA
CAC TCGGAGC CC CGGC CCAATGAGCACAAC TGC TCGGC TAGCAGGAGGATCC GC CCAGAG TCAC
TCCA
GGGTGCTGACCACCGGCCCTACACCTTCTTCATTTCCCCGGGGAGCAGAGACCCAGCGGGGAGTTACC
ATCTGAACCTCTCCAGCCACTTCCGCTGGTCGGCGCTGCAGGTGTCCGTGGGCCTGTACACGTCCCTG
TGCCAG TAC T TCAGCGAGGAGGACATGG TG TGGC GGACAGAGGGGC TGC TGC CC C TGGAGGAGACC
TC
GCCCCGCCAGGCCGTCTGCCTCACCCGCCACCTCACCGCCTTCGGCGCCAGCCTCTTCGTGCCCCCAA
GC CATG TC CGC T TTGTGT TTCC TGAGCC GACAGC GGATGTAAAC TACATC GTCATGC TGACATG
TGC T
GTGTGC C TGG TGAC C TACATGG TCATGGCC GC CATC C TGCACAAGC TGGACCAG TTGGATGC
CAGCC G
GGGCCGCGCCATCCCTTTCTGTGGGCAGCGGGGCCGCTTCAAGTACGAGATCCTCGTCAAGACAGGCT
GGGGCCGGGGCTCAGGTACCACGGCCCACGTGGGCATCATGCTGTATGGGGTGGACAGCCGGAGCGGC
CACC GGCACC TGGACGGC GACAGAGC C T TC CACC GCAACAGC C TGGACATC T TC CGGATC GC
CACCC C
GCACAGCC TGGG TAGC GTGTGGAAGATC CGAG TG TGGCAC GACAACAAAGGGC TCAGC CC TGCC TGG
T
TC C TGCAGCACG TCATCG TCAGGGAC C TGCAGAC GGCACGCAGC GC C T TC TTCC TGGTCAATGAC
TGG
C T TTCGGTGGAGAC GGAGGC CAAC GGGGGC C TGG TGGAGAAGGAGG TGC TGGCC GC GAGC
GACGCAGC
CC TT TTGCGC TTCCGGCGCC TGCTGGTGGC TGAGCTGCAGCGTGGC TTCT TTGACAAGCACATC TGGC
TCTCCATATGGGACCGGCCGCCTCGTAGCCGTTTCACTCGCATCCAGAGGGCCACCTGCTGCGTTCTC
CTCATCTGCCTCTTCCTGGGCGCCAACGCCGTGTGGTACGGGGCTGTTGGCGACTCTGCCTACAGCAC
GGGGCATGTGTCCAGGCTGAGCCCGCTGAGCGTCGACACAGTCGCTGTTGGCCTGGTGTCCAGCGTGG
TTGTCTATCCCGTC TACC TGGCCATCCT TT TTCTCT TCCGGATGTCCCGGAGCAAGGTGGCTGGGAGC
CC GAGC CC CACACC TGCC GGGCAGCAGG TGC TGGACATCGACAGC TGC C TGGAC TC GTCC GTGC
TGGA
CAGCTCCTTCCTCACGTTCTCAGGCCTCCACGCTGAGCAGGCCTTTGTTGGACAGATGAAGAGTGACT
TG TT TC TGGATGAT TC TAAGAG TC TGGTGTGC TGGC CC TC CGGC GAGGGAAC GC TCAG
TTGGCC GGAC
C TGC TCAG TGAC CC GTCCAT TG TGGG TAGCAATC TGCGGCAGC TGGCACGGGGC
CAGGCGGGCCATGG
.. GC TGGGCCCAGAGGAGGACGGC TTCTCCCTGGCCAGCCCC TACTCGCC TGCCAAATCC TTCTCAGCAT
CAGATGAAGACC TGATCCAGCAGG TC C T TGCC GAGGGGGTCAGCAGCC CAGC CC C TAC CCAAGACAC
C
CACATGGAAACGGACCTGCTCAGCAGCCTGTCCAGCACTCCTGGGGAGAAGACAGAGACGCTGGCGCT
GCAGAGGCTGGGGGAGCTGGGGCCACCCAGCCCAGGCCTGAACTGGGAACAGCCCCAGGCAGCGAGGC

TGTCCAGGACAGGACTGGTGGAGGGTCTGCGGAAGCGCCTGCTGCCGGCCTGGTGTGCCTCCCTGGCC
CACGGGCTCAGCCTGCTCCTGGTGGCTGTGGCTGTGGCTGTCTCAGGGTGGGTGGGTGCGAGCTTCCC
CCCGGGCGTGAGTGTTGCGTGGCTCCTGTCCAGCAGCGCCAGCTTCCTGGCCTCATTCCTCGGCTGGG
AGCCACTGAAGGTCTTGCTGGAAGCCCTGTACTTCTCACTGGTGGCCAAGCGGCTGCACCCGGATGAA
GATGACACCCTGGTAGAGAGCCCGGCTGTGACGCCTGTGAGCGCACGTGTGCCCCGCGTACGGCCACC
CCAC GGC T TTGCAC TC TTCC TGGC CAAGGAAGAAGC CC GCAAGG TCAAGAGGC TACATGGCATGC
TGC
GGAGCCTCCTGGTGTACATGCTTTTTCTGCTGGTGACCCTGCTGGCCAGCTATGGGGATGCCTCATGC
CATGGGCACGCCTACCGTCTGCAAAGCGCCATCAAGCAGGAGCTGCACAGCCGGGCCTTCCTGGCCAT
CACGCGGTCTGAGGAGCTCTGGCCATGGATGGCCCACGTGCTGCTGCCCTACGTCCACGGGAACCAGT
CCAGCC CAGAGC TGGGGC CC CCAC GGC TGC GGCAGG TGCGGC TGCAGGAAGCAC TC TACC
CAGACCC T
CCCGGCCCCAGGGTCCACACGTGCTCGGCCGCAGGAGGCTTCAGCACCAGCGATTACGACGTTGGCTG
GGAGAG TC C TCACAATGGC TCGGGGACG TGGGCC TATTCAGC GC CGGATC TGC TGGGGGCATGG TCC
T
GGGGCTCCTGTGCCGTGTATGACAGCGGGGGCTACGTGCAGGAGCTGGGCCTGAGCCTGGAGGAGAGC
CGCGACCGGCTGCGCTTCCTGCAGCTGCACAACTGGCTGGACAACAGGAGCCGCGCTGTGTTCCTGGA
GC TCACGCGC TACAGCCCGGCCGTGGGGCTGCACGCCGCCGTCACGCTGCGCCTCGAGTTCCCGGCGG
CCGGCCGCGCCCTGGCCGCCCTCAGCGTCCGCCCCTTTGCGCTGCGCCGCCTCAGCGCGGGCCTCTCG
CTGCCTCTGCTCACCTCGGTGTGCCTGCTGCTGTTCGCCGTGCACTTCGCCGTGGCCGAGGCCCGTAC
TTGGCACAGGGAAGGGCGCTGGCGCGTGCTGCGGCTCGGAGCCTGGGCGCGGTGGCTGCTGGTGGCGC
TGACGGCGGCCACGGCACTGGTACGCCTCGCCCAGCTGGGTGCCGCTGACCGCCAGTGGACCCGTTTC
GTGCGCGGCCGCCCGCGCCGCTTCACTAGCTTCGACCAGGTGGCGCAGCTGAGCTCCGCAGCCCGTGG
CC TGGCGGCC TCGC TGCTCTTCCTGC TTTTGGTCAAGGCTGCCCAGCAGC TACGCTTCGTGCGCCAGT
GGTCCGTCTTTGGCAAGACATTATGCCGAGCTCTGCCAGAGCTCCTGGGGGTCACCTTGGGCCTGGTG
GTGCTCGGGGTAGCCTACGCCCAGCTGGCCATCCTGCTCGTGTCTTCCTGTGTGGACTCCCTCTGGAG
CGTGGCCCAGGCCCTGTTGGTGCTGTGCCCTGGGACTGGGCTCTCTACCCTGTGTCCTGCCGAGTCCT
GGCACCTGTCACCCCTGCTGTGTGTGGGGCTCTGGGCACTGCGGCTGTGGGGCGCCCTACGGCTGGGG
GC TGTTATTC TCCGCTGGCGCTACCACGCC TTGCGTGGAGAGCTGTACCGGCCGGCCTGGGAGCCCCA
GGAC TACGAGATGG TGGAGT TG TTCC TGCGCAGGC TGC GC C TC TGGATGGGC C TCAGCAAGG
TCAAGG
AGTTCCGCCACAAAGTCCGCTTTGAAGGGATGGAGCCGCTGCCCTCTCGCTCCTCCAGGGGCTCCAAG
GTATCCCCGGATGTGCCCCCACCCAGCGCTGGCTCCGATGCCTCGCACCCCTCCACCTCCTCCAGCCA
GC TGGATGGGCTGAGCGTGAGCCTGGGCCGGC TGGGGACAAGGTGTGAGCCTGAGCCC TCCCGCCTCC
AAGC CG TG TTCGAGGC CC TGC TCACC CAGT TTGACC GAC TCAAC CAGGCCACAGAGGACG TC
TACCAG
CTGGAGCAGCAGCTGCACAGCCTGCAAGGCCGCAGGAGCAGCCGGGCGCCCGCCGGATCTTCCCGTGG
CCCATCCCCGGGCCTGCGGCCAGCACTGCCCAGCCGCCTTGCCCGGGCCAGTCGGGGTGTGGACCTGG
CCAC TGGC CC CAGCAGGACACC CC TTCGGGCCAAGAACAAGG TC CACC CCAGCAGCAC TTAGT C CT
CC
TTCCIGGCGGGGGIGGGCCGTGGAGTCGGAGTGGACACCGCTCAGTATTACTITCTGCCGCTGICAAG
GCCGAGGGCCAGGCAGAATGGCTGCACGTAGGITCCCCAGAGAGCAGGCAGGGGCATCTGICTGICTG
TGGGCTICAGCACTITAAAGAGGCTGIGTGGCCAACCAGGACCCAGGGICCCCTCCCCAGCTCCCITG
GGAAGGACACAGCAGTATTGGACGGITTCTAGCCICTGAGATGCTAATTTATTICCCCGAGTCCTCAG
GTACAGCGGGCTGTGCCCGGCCCCACCCCCIGGGCAGATGICCCCCACTGCTAAGGCTGCTGGCTICA
GGGAGGGITAGCCTGCACCGCCGCCACCCTGCCCCTAAGTTATTACCICTCCAGTTCCTACCGTACTC
CCTGCACCGICTCACTGIGTGICTCGTGICAGTAATTTATATGGIGTTAAAATGIGTATATTTITGTA
TGTCACTATTTTCACTAGGGCTGAGGGGCCTGCGCCCAGAGCTGGCCTCCCCCAACACCTGCTGCGCT
TGGTAGGTGTGGTGGCGTTATGGCAGCCCGGCTGCTGCTTGGATGCGAGCTTGGCCTTGGGCCGGTGC

TGGGGGCACAGCTGTCTGCCAGGCACTCTCATCACCCCAGAGGCCTTGTCATCCTCCCTTGCCCCAGG
CCAGGTAGCAAGAGAGCAGCGCCCAGGCCTGCTGGCATCAGGTCTGGGCAAGTAGCAGGACTAGGCAT
GICAGAGGACCCCAGGGIGGITAGAGGAAAAGACTCCTCCTGGGGGCTGGCTCCCAGGGIGGAGGAAG
GTGACTGTGTGTGTGTGTGTGTGCGCGCGCGCACGCGCGAGTGTGCTGTATGGCCCAGGCAGCCTCAA
GGCCCTCGGAGCTGGCTGTGCCTGCTICTGIGTACCACTICTGIGGGCATGGCCGCTICTAGAACGGG
TGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAG
CCTIGTCCTAATAAAATTAAGTTGCATCATTITGICTGACTAGGIGTCCTICTATAATATTATGGGGT
GGAGGGGGGIGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCIGTAGGGCCTGCGGGGICTATTGG
GAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGAT
TCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGT
TT TT TIGGTAGAGACGGGGT TTCACCATAT TGGCCAGGCTGGICTCCAACTCCTAATCTCAGGTGATC
TACCCACCTIGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTICCCTGICCITTA
AC TATAAC GGT C CTAAGGTAGC GAAGGC TCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAG
TC CC CGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAAC
TGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGC
AGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTG
GTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTG
CAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTA
AGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCT
GGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCT
GCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTC
GGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCC
TGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGC
CTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGC
GGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGAAGGACGCGGCGCTCGGGAGAGC
GGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCC
ACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGG
TTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAG
CTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAG
CCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGATCCGGAGGCGGCGGCACGGG
CGGCGGCAGCGGCGGCATGGTGAACTCCAGTCGCGTGCAGCCTCAGCAGCCCGGGGACGCCAAGCGGC
CGCCCGCGCCCCGCGCGCCGGACCCGGGCCGGCTGATGGCTGGCTGCGCGGCCGTGGGCGCCAGCCTC
GCCGCCCCGGGCGGCCTCTGCGAGCAGCGGGGCCTGGAGATCGAGATGCAGCGCATCCGGCAGGCGGC
CGCGCGGGACCCCCCGGCCGGAGCCGCGGCCTCCCCTTCTCCTCCGCTCTCGTCGTGCTCCCGGCAGG
CGTGGAGCCGCGATAACCCCGGCT TCGAGGCCGAGGAGGAGGAGGAGGAGGTGGAAGGGGAAGAAGGC
GGAATGGIGGIGGAGATGGACGTAGAGIGGCGCCCGGGCAGCCGGAGGICGGCCGCCTCCTCGGCCGT
GAGCTCCGTGGGCGCGCGGAGCCGGGGGCTTGGGGGCTACCACGGCGCGGGCCACCCGAGCGGGAGGC
GGCGCCGGCGAGAGGACCAGGGCCCGCCGTGCCCCAGCCCAGTCGGCGGCGGGGACCCGCTGCATCGC
CACCTCCCCCTGGAAGGGCAGCCGCCCCGAGTGGCCTGGGCGGAGAGGCTGGTTCGCGGGCTGCGAGG
TCTCTGGGGAACAAGACTCATGGAGGAAAGCAGCACTAACCGAGAGAAATACCTTAAAAGTGTTTTAC
GGGAACTGGTCACATACCTCCTTTTTCTCATAGTCTTGTGCATCTTGACCTACGGCATGATGAGCTCC
AATGIGTACTACTACACCCGGATGATGICACAGCTCTICCTAGACACCCCCGTGICCAAAACGGAGAA
AACTAACTITAAAACTCTGICTICCATGGAAGACTICTGGAAGTICACAGAAGGCTCCITATTGGATG

GGCT GTACTGGAAGAT GCAGCCCAGCAACCAGACTGAAGCTGACAACCGAAGTT TCATCT TCTATGAG
AACCTGCTGTTAGGGGITCCACGAATACGGCAACTCCGAGICAGAAATGGATCCTGCTCTATCCCCCA
GGACTTGAGAGATGAAATTAAAGAGTGCTATGATGICTACTCTGICAGTAGTGAAGATAGGGCTCCCT
TT GGGCCCCGAAAT GGAACCGCTT GGATCTACACAAGT GAAAAAGACT TGAATGGTAGTAGCCACTGG
GGAATCAT TGCAACTTATAGTGGAGCTGGCTATTATCT GGAT TT GTCAAGAACAAGAGAGGAAACAGC
TGCACAAGTT GCTAGCCTCAAGAAAAAT GT CT GGCT GGACCGAGGAACCAGGGCAACT TT TATT GACT
TCTCAGIGTACAACGCCAACATTAACCTGITCTGIGTGGICAGGITATTGGITGAATTCCCAGCAACA
GGTGGT GT GATTCCATCT TGGCAATT TCAGCCTT TAAAGCTGATCCGATATGTCACAACT TT TGATT T
CT TCCT GGCAGCCT GT GAGATTATCT TT TGIT TCTITATCTITTACTATGIGGIGGAAGAGATATTGG
AAATTCGCATTCACAAACTACACTATTICAGGAGTTICTGGAATTGICTGGATGTTGTGATCGTTGTG
CT GTCAGT GGTAGCTATAGGAATTAACATATACAGAACATCAAATGIGGAGGIGCTACTACAGTTICT
GGAAGATCAAAATACT TTCCCCAACT TT GAGCATCT GGCATATT GGCAGATACAGT TCAACAATATAG
CT GCTGTCACAGTATT TT TT GT CT GGAT TAAGCTCT TCAAAT TCATCAAT TT TAACAGGACCAT
GAGC
CAGCTCTCGACAACCATGICTCGATGTGCCAAAGACCTGITTGGCTITGCTATTATGITCTICATTAT
TTTCCTAGCGTATGCTCAGTTGGCATACCTTGTCTTTGGCACTCAGGTCGATGACTTCAGTACTTTCC
AAGAGT GTATCT TCACTCAATTCCGTATCATT TT GGGCGATATCAACT TT GCAGAGAT TGAGGAAGCT
AATCGAGT TT TGGGACCAAT TTAT TTCACTACAT TT GT GT TCTT TATGTICT TCAT TCTT TT
GAATAT
GT TT TT GGCTATCATCAATGATACTTACTCTGAAGT GAAATCTGACTT GGCACAGCAGAAAGCT GAAA
TGGAACTCTCAGATCT TATCAGAAAGGGCTACCATAAAGCTT TGGICAAACTAAAACT G
T
ACCGTGGATGACAT TICAGAGAGICT GCGGCAAGGAGGAGGCAAGT TAAACT TT GACGAACT TCGACA
AGATCTCAAAGGGAAGGGCCATACTGATGCAGAGATTGAGGCAATATTCACAAAGTACGACCAAGATG
GAGACCAAGAACTGACCGAACATGAACATCAGCAGATGAGAGACGACTTGGAGAAAGAGAGGGAGGAC
CT GGAT TT GGAT CACAGT TCTT TACCACGTCCCATGAGCAGCCGAAGT TICCCTCGAAGCCT GGATGA
CTCT GAGGAGGATGACGATGAAGATAGCGGACATAGCTCCAGAAGGAGGGGAAGCATT TCTAGT GGCG
TT TCTTACGAAGAGTT TCAAGTCCIGGT GAGACGAGTGGACCGGAT GGAGCATTCCATCGGCAGCATA
GT GTCCAAGATT GACGCCGT GATCGT GAAGCTAGAGAT TATGGAGCGAGCCAAACT GAAGAGGAGGGA
GGIGCTGGGAAGGCTGTTGGATGGGGIGGCCGAGGATGAAAGGCTGGGICGTGACAGTGAAATCCATA
GGGAACAGAT GGAACGGCTAGTACGT GAAGAGTT GGAACGCT GGGAATCCGATGAT GCAGCT TCCCAG
ATCAGICATGGITTAGGCACGCCAGIGGGACTAAATGGICAACCTCGCCCCAGAAGCTCCCGCCCATC
TTCCTCCCAATCTACAGAAGGCATGGAAGGIGCAGGIGGAAATGGGAGTTCTAATGICCACGTATGAT
TCTAGAGTCGACCTGCAGAAGCTTGCCTCGAGCCTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGC
CCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGA
AATTGCATCGCATTGTC TGAGTAGGTGTCATTC TAT TC TGGGGGGTGGGG TGGGGCAGGACAGCAAGG
GGGAGGAT TGGGAAGACAATAGCAGGCATGC TGGGGATGC GG TGGGC TC TATGGTAAC TATAACGGTC
CTAAGGTAGCGAAGTCGACCGAATCGTT GTCCCT TGTCACAGCCAT TGAGAATT TT GGCAGGGAGCAT
GTTCTTAGAGCATTTTTAGGCTCTGCGGGACATAACAGCTCTGCCTCAGAGCACATGCCTTTCTCAGC
TCCT GAAAGCCACT GATCAAAT TGGAACAT TT TGTACCTTAGGGAT GAGGATATCAACTCTCCCAGCC
ACTTAGAGGGATAAAT GT GATGAT GCAT TCAATT GT GACTACATCT GATCCCAACT GT TGCT TCAGCT

GCTCTCCTATAGCACATGGCGGGAGGCGTGCATCCCAGTAGCTACCTCCCCACTTTTGGGGAGATGTG
GITCCATCCATGAAACCIGGGTACCCGCCTACCAGGICCIGGCCTATCAGGIGGCAGGGICTGGICAA
AGAAGGGCAT GT GT GGICTICAGCAAGGGAGACAGGACGGIGGT GCAGAGCGICTAGACCCTCAGGGC
AAGICTCCCCCACACCTGCTCCCGGGGCAGTTGICITTGTGACCTCCCATCCCCCTCTGITTCATCCT
CTATAAAATGAGGGGCTGAGCCCCAAAATAACAGGCTICT TT GCCATGAT GCAAAACT GCTGAATCT T

TCTTTCTGACACACAAGGCATCGAGCAGCCTCTGAAAGAACCAAAGCCACTAGCAGGCTTCCTGACTT
GGGT TT GTAGGTACTGAATACTCCCT TGAAAAATAAAAACATAGAGGCACTT TTCTCCTGGCTGTTTA
TTACAGAACGAAGAAAAAACACACTGGCTT GAAACAGACGCCAGAT TT CAAATGTAGAGGTGAAATAC
GAGGTGGCAATTAAAATGTGATTACAGAAAGTCTGGACACTGAGAAAAGTTTACAGGACAGTGGGTGT
GGGTTTTCTATAACAGACACTTAAATATACATGACGATAATTGCAGATAGAAACCATCAAAGACAAAC
CCCAAATCAACTAATAAT GT TTACAGAT GT TCCCCCCCAAACCACAGAGCCT TACATCAAAACAAATA
CT GAAAGGCT TTAAACCAGGAACAGCTCGCCT TAACCCCACGAGGGTGCACACAAGCT GGGCTT TTTC
TCTCGGTCTGAATGGTAAAGGGAGGAGGATACTCTAGCTCCTCCAGGTGGATTGCTGAGACAGGGCTC
GGCTCACACACTGTCTCTGCGCCTCTCCCAAATCTGGAGAACTCTCCCAGCCTCCTGGTAAAGTGTCT
CT GT GGGGCACT TAACGATAAAACAGCT TCTGCT GTAAAGCTCATTAGGAAAGAGCTAGCGGAGACT G
AAAGGTTCGCAAAAGAGATTAAGAATCACACAAGGCAATAGGATTTTTAGTGAACATAGAAATAAATG
GCCAAGTGGTTTTCTATTTGGCATTTGTCAACTTGCACAACAACTCTTGGTCATATCCACATTGCTCA
TT GCAT TAAAACCATAAGCGACTCAGCCACCTAGCT TAACAAGGTATCACTGGAGCAAACAACACGGT
CT GCATAT TT GTAACATT GTATAATAAACACAAAACAATGCATAGTAAACACAACT CTACTGAAACAA
AAGCCGTCGCTT TATT TACAAAGTCACAAAAT GAAGTATAAATACT TCTGTCAT TAAT GT TTAGGAAA
ACCATT TACAAAAT TT TCAAATAT GTACACGTAGCT TGAAAAATCACCAGCT TTCCAT TT TGTCACAG
GTAGAGAGAGGGATAAGCATGGGCTGACAACACCACTCAAATTGTAACGGGAGACAACTGCGGGTATG
GATCGACACCACTTCCTAGAGTGATGTCACCATGGGGGTTTCTATGGGCATCCTGCTCAGATTTAAAG
TGCCCCAGCATCCTGGGTGACTTGCCCAGAATTCTGGGCTGTGGCATTTTGAGCAGCAGCATGCTGTT
CCAAAATGTCGTCGATCAGCCTCAAGTTGCACACCCAGTCTTCATCTGGGCTCACACAGGAGCCTTTC
AAGAGAGCTTCAATGAAATCTACCTCATTGCAGTCAGGTGACGAAATCAGATCATTTAGTGGGGGTTG
GGGCTGGCGCAAAAAGTCGGCAGGTGGCAGCTCAGGGGGAATATCCGTTCTGTCGAACGGACCTGGGA
ACTGGCTGGCAGCAACGGCAGAAGCAGCAGCAGCGGTGGCAGCAGCAGCCACATAGCTTGGTGGCTCG
ATGCCCTGTATGGGGCTCAGGGGACTAAAGCTGGCCATACCCTGCTGGAGGAACTTGGTGGTGTTTGC
TACAGGCACCGGGCCCTGTACCGGGCTCTGCCTGAGGCTCTGGCTGCCCAGCAGGCTGAAGCTGGGGT
TGTTGGCCAGGGGCACTTGTGTTCCCATCGCAGCGGGCACTTGTGCCTCCCAATCAGATGGCCTCTGA
AGGCAGGCCTGGCCAGAAGGTGAGTGCTGCTGAACGCTATTATCCACTTGGCTGAGGGGTGTTTTCCC
CGAAACTGCTGTGGTCACAGCTGCTGCCGCTGTGACCCATGCAGCATTGTTGAACGCAGTGGGCATTC
TT GGCACACTAGGCCGTCTGAGCT GGTGGGGACTCAAGGACT GGGT GCCCAGGGAGCT GGGACAGAAC
CCAGGCAGGGGCACTTCTGGTGGGGTGGCCTTGGGGCTCTGCATATGCTGGCAGACAGAGTCAAGTCT
GCCCAGGGGAGTCTGGCCTGAGTGTGAGAGGATGGGACACTGGGGGCTGGAGGTGAAAATTCCTTGCC
GCTTCCCCAGAGTTGGTGAGATCACTCCCATGCCCTCGCAGCTCTGGTGCCTGGTGAGTGGGATCATT
CCTGGACTCAGATTGTTCTGAAGAAGCCCAGTTCTGGGTGGCATCAAGTGCTTGCTAGATGGGGGGCT
TGCCTT GATCCGGCTACACT TGGAGGTGACTT GT TCTT GGACGGCTACATACAGAAAGAGAGAAGTGG
GGATGAGTTCCAAAGGCATCCTCGACTTCGGCTGTGGCCACCGGAGGGTAGCTCCTGGCCCAACACGG
ACTTCTCACCTCCCGCCCTTGGCTCTCTACTGAGCTCCCCCCTGCTCCCCAATTCCTCGCCATTCCCC
TCATTTCTCTGCCCTCAGCCTGGACTGCAGTTCTTCTGGGAAGCTGCCCCAACTCCCTAGGTCTGTGC
TCACCAAGAGCAGATCACACTGGACT GAAATGCCAGCT GATT TGTCTCTTCAAGAAAATT GGAAGCTC
CT GGAGGTCAGGGTCCAT GI CT GCTT TTACACTCAGTGCTCT GTAT GCAGGCCT GGCACT GCCCACCC
TT TGACAGGT GGTGCATATT TT GTAGAAGGAAGGAAGGGGCCAGGT GGGGTGGGCT GGGCTGGT GGCG
GGAGCTAGCTCAGCCTCT TAGATTCTCTACCCGATGGATGTGACCT GGGACAGCAAGT GAGT GT GGT G
AGTGAGTGCAGACGGTGCTTTGTTCCCCTCTTGTCTCATAGCCTAGATGGCCTCTGAGCCCAGATCTG
GGGCTCAGACAACATTTGTTCAACTGAACGGTAATGGGTTTCCTTTCTGAAGGCTGAAATCTGGGAGC

TGACATTCTGGACTCCCTGAGTTCTGAAGAGCCTGGGGATGGAGAGACACGGAGCAGAAGATGGAAGG
TAGAGTCCCAGGTGCCTAAGATGGGGAATACATCTCCCCTCATTGTCATGAGAGTCCACTCTAGCTGA
TATCTACTGTGGCCAATATCTACCGGTACT TT TT TGGGGTGGACACTGAGTCATGCAGCAGTCT TATG
GT TTACCCAAGGTCAGGTAGGGGAGACAGTGCAGTCAGAGCACAAGCCCAGTGTGTCTGACCCACCCA
AGAATCCATGCTCGTATCTACAAAAATGATTTTTTCTCTTGTAATGGTGCCTAGGTTCTTTTATTATC
ATGGCATGTGTATGTT TT TCAACTAGGT TACAATCTGGCCTTATAAGGTTAACCTCCTGGAGGCCACC
AGCCTTCCTGAAACTTGTCTGTGCTGTCCCTGCAACTGGAGTGTGCCTGATGTGGCACTCCAGCCTGG
ACAAGTGGGACACAGACTCCGCTGTTATCAGGCCCAAAGATGTCTTCCATAAGACCAGAAGAGCAATG
GTGTAGAGGTGTCATGGGCTACAATAAAGATGCTGACCTCCTGTCTGAGGGCAAGCAGCCTCTTCTGG
CCCTCAGACAAATGCTGAGTGTTCCCAAGACTACCCTCGGCCTGGTCCAATCTCATCCCACTGGTGCG
TAAGGGTTGCTGAACTCATGACTTCTTGGCTAGCCTGCAACCTCCACGGAGTGGGAACTACATCAGGC
AT TT TGCTAACTGCTGTATCCTAGGCCAATAAATGT TGATCACATT TATAGCTGCCATGGTAGGGTGG
GGACCCCTGCTATCTATCTGTGGAGGCTCTGGGAGCCCCTGACACAAACTTTCTGAAGCAGAGCCTCC
CCAACCCCTTTTCCATTCCCTATACCTGACAGATGGCCCAGGAACCCATTAGAAATGGAAGGTCACTG
CAGCAGTATGTGAATGTGCGTGTGGGAGAAGGGCAGGATCAGAGCCCTGGGGGTGTGGCAGCCCCCAA
GTGATTCTAATCCAGATCCTAGGGTTGTTTCCCTGTCCCATTGAAATAGCTGCTTTAAGGGGCCTGAC
TCAGGGAAATCAGTCTCT TGAATTAAGTGGTGAT TT TGGAGTCATT TAGACCAGGCCT TCAATTGGGA
TCCACTAGTTCTAGAGCGGCCGGGCCCAGGGAACCCCGCAGGCGGGGGCGGCCAGTTTCCCGGGTTCG
GCTTTACGTCACGCGAGGGCGGCAGGGAGGACGGAATGGCGGGGTTTGGGGTGGGTCCCTCCTCGGGG
GAGCCCTGGGAAAAGAGGACTGCGTGTGGGAAGAGAAGGTGGAAATGGCGTTTTGGTTGACATGTGCC
GCCTGCGAGCGTGCTGCGGGGAGGGGCCGAGGGCAGATTCGGGAATGATGGCGCGGGGTGGGGGCGTG
GGGGCTTTCTCGGGAGAGGCCCTTCCCTGGAAGTTTGGGGTGCGATGGTGAGGTTCTCGGGGCACCTC
TGGAGGGGCCTCGGCACGGAAAGCGACCACCTGGGAGGGCGTGTGGGGACCAGGTTTTGCCTTTAGTT
TTGCACACACTGTAGTTCATCTTTATGGAGATGCTCATGGCCTCATTGAAGCCCCACTACAGCTCTGG
TAGCGGTAACCATGCGTATTTGACACACGAAGGAACTAGGGAAAAGGCATTAGGTCATTTCAAGCCGA
AATTCACATGTGCTAGAATCCAGATTCCATGCTGACCGATGCCCCAGGATATAGAAAATGAGAATCTG
GTCCTTACCTTCAAGAACATTCTTAACCGTAATCAGCCTCTGGTATCTTAGCTCCACCCTCACTGGTT
TT TI CT TGTT TGTTGAACCGGCCAAGCTGCTGGCCTCCCTCCTCAACCGT TCTGATCATGCT TGCTAA
AATAGTCAAAACCCCGGCCAGT TAAATATGCT TTAGCCTGCT TTAT TATGAT TATT TT TGTTGT TTTG
GCAATGACCTGGTTACCTGTTGTTTCTCCCACTAAAACTTTTTAAGGGCAGGAATCACCGCCGTAACT
CTAGCACTTAGCACAGTACTTGGCTTGTAAGAGGTCCTCGATGATGGTTTGTTGAATGAATACATTAA
ATAATTAACCACTTGAACCCTAAGAAAGAAGCGATTCTATTTCATATTAGGCATTGTAATGACTTAAG
GTAAAGAGCAGTGCTATTAACGGAGTCTAACTGGGAATCCAGCTTGTTTGGGCTATTTACTAGTTGTG
TGGCTGTGGGCAACTTACTTCACCTCTCTGGGCTTAAGTCATTTTATGTATATCTGAGGTGCTGGCTA
CCTCTTGGAGTTATTGAGAGGATTATAAGACAGTCTATGTGAATCAGCAACCCTTGCATGGCCCCTGG
CGGGGAACAGTAATAATAGCCATCATCATGTT TACT TACATAGTCCTAAT TAGTCT TCAAAACAGCCC
TGTAGCAATGGTATGATTAT TACCAT TT TACAGATGAGGAACCT TTGAAGCCTCAGAGAGGCTAACAG
ACATACCCTAGGTCATACAGTTATTAAGAGAAGGAGCTCTGTCTCGAACCTAGCTCTCTCTCTCTCGA
GTAATACCAGTTAAAAAATAGGCTACAAATAGGTACTCAAAAAAATGGTAGTGGCTGT TGTT TT TAT T
CAGT TGCTGAGGAAAAAATGTTGATT TT TCATCTCTAAACATCAACTTACTTAATTCTGCCAAT TTCT
TTTTTTTGAGACAGGGTCTCACTCTGTCACCTAGGATGGAGTGCAGTGGCACAATCACTGCTCACTGC
AGCCTCGACTTCCCGGGCTCGGGTGATTCTCCCCAGGCTCAGGGGATTCTCCCACTTCAGCCTCCCAA
GTAGCTGGGACTACAGGTGCGCACCACCATCCCTGGCTAATATTTGTACTTTATTTTATTTATTTATT

TATT TATT TT TT GAGATGGAGT TTCGCTCT TGTT GCCCAAAT GAAT TGCCTCTTAT TTAATT
TCGTCT
GATGATACAT TT TGTT TT TATT TT GTAAAAAATTAT TT TT TT TCTT TT TGGAGACAGGGTCT
TGCTCT
GTTGCCCAGGCTGGTCACAAACTCCTGACCTCAAGCAATCCTCCTGCCTTAGCCTCCCAAAATGCTGG
GATTACAGGCGT GACGACCTCGCCCGGCCT TGTATTAT GATACATT TT GAACAACTACAAGTAGACT T
GGTATAATGAACCTGCACGTACCCATTGCCAAGTTCTGACAACTGTCTGTCTATAGCCAATTATGCAT
TTCT TAAATTAGAACCCCCCCAATATACCCAAATATATATATAT GT GT GCATATATATAGTAAGTTGT
AACAAAGT TGTGAATTCATACCTGAAGTATCTCAAGTGAT GCAAGT TT TATGAATT TT TGTT TATGCC
TTTTGGGAAGAGTTGTATTGACAAATTTTTTATGCTTAAAGTAAACCATAAATCAAAAAAATAAAATC
TAGGAT GCAATAAAACAAAACAACTTCT TGACATAAGTAT GGTATGTAAATCTGTT TT GATT GGAAAT
CAATTTGTTATATTGCCAGAATTCCTGTTTTAGAATACATCTCTGCTGATCTGTCTGTATTCTTAGAC
TGCATATCTGGGAT GAACTCTGGGCAGAAT TCACAT GGGCTTCCTT TGAAATAAACAAGACT TT TCAA
ATTCTTAGTCGATCTGCAGAACCTGTAGCCAGGCACTGAACCATTTTGATAGATGCAGTAATCGTTGC
AAGT GTATAT TTCAAGGGAGTTCT GGCT GGGTCCTAGT TTAT GCTT GT GGCAGAAGCAGT GAGTAACT
GGGAGGAAGT TGGT GAGTAAGCTTCAAGGAAGAAGTCATT TT TAGTACTCTGGATCTTCCTGAT TTTA
AAGCACTACAAAATGGTGCATTTTCATTCTTGTCAAGTGATAACAGATATATTCTGATGAGCCTGAAA
TGAATATATATT GTATCATT TT TATAATATCTAGCAAGGT TT GTAT TT TCCTAGAACT TGAACTAAAT
TTCAGT TCATAAAATITATAAAATACTTAGTT GT TGTAAAATAT TT TT GGAATGITCACATAGGTGAC
ACACAAATGTCCCATTTTCATTCTTTCTATAGTAAATATGTTCTGATATGTGAAGGTTTAGCAGATGC
ATCAGCAT TTAATCCTAGAGGATCTGGCATAATCTT TTCCCCCAAGAATAGAAATT TT TI CT GCTTAT
GAAAGTAGTACATGTTTCTTTAAAAACAAATCAATATTGACTTCTGCCTGCTGTATAGCACTATGCCT
CCACCTGGCCATGACCAGGGGCATGTCCTGGTCCACCTACCTGAAAATGTTTGCAACCAGCCTCCTGG
CCAT GT GCACAGGGGCTGAAGT TGTCCCACAGGTAT TACGGGCCAACCTGACAATACATGAAGT TCCA
CCAAAGTCTGAGAACTCAGAACTGAGCT TT GGGGACTGAAAGACAGCACAAACCTCAAAT TTCTCAGC
ACTGGAAACCTCAAAATATAACTGAATTCCATAAATAAGATTTTAAGTCTTAAATATGTATTTTTAAA
TGTATTAAAAGTCAAGCTGCTTGTATTTAAGCACCTAATACAATGCTTAGGTTGTAAAAGGAGATGCT
CAATAGGTACTAACTGATATATTGAGATTTAATTATGGTTTGACCAATATTTATTGGAAACCGCCAAA
GCTTAAATCATCAGCT TCTT GAAT GT GATT TGAAAGGTAATT TAGTAT TGAATAGCAT GT GAGCTAGA
GTAT TTCATTCT TTCT GGTT TATT TCTTCAAATAGACT TT GAATATAATGGT GAAT GGGTAT TATAAA

TTAACTAATAAAAATGACATTGAAAATGAAAAAATATATATATTAAAGTGTAGAAAGTGACCAGGCGT
GGTGGCTCACACCTGTAATCCAAGCACCTTGGGAGGCTGAGGCAGGAGGATCTCTTGATCCCAGGAGT
TCAAGACCAGCCTGGGCAACATAGCGAGACTTCGTCTCT
GAGAGAGAAAAAAATTT
TT TT TATT TAAAAAAAGT GTAGAAAGTGTCAAGACCCCACTTCT TACCAT TATT TGGTATAT TTCTCT
ATACCCACCCACCCTTCCTCCTTACTCCCTCCCTCCCTTCCCAATCTTTTTATCTTTTTGTATTCTGA
TTTTTTGTTTGTATATTTTGCTTTAATTTAATGTATCCTTTAAAAATTTCCCATACATTTTATATGTA
TATATAAAAACGCATGCTGCCAAAGATAATTTATAAGAAAGACCATTGAATTTTTTTAAAAGTGATAT
ATATTCATTGAAAAAAATTTAGAATATATAGCAAAGCAATAAAGAACTAAATAAAATTGCTGTAACTC
CTCTTTCAAAGATAAGTGCTTTTATGATTTTGTTGTATTTTTTTCTGTATATAGGTACATATATAGTA
TT TATAAAGCTGTACTCATAGTACAT TT TCACATCACAGGTACCATATCAGT GT TATTAAATAT TTT G
TATGCCAGGGGCTAGACATACCAAGACAACCAATAT GT GGTTCTACTTAAATAATATTAGAGTATCT T
TTATGATGACACTTCATGAGTTGACTATAATAATCTTAGACTTCTAAGAGTTTGGGTTTTCAAAAGAT
CACTTAGCTTTTTTGGGTGATTTTTCCCCCTTACTGTGAGATGAGAGAGGCTGTTTGGATTTGGGATT
GGGGTAGCGGGGACAGCAACTTTTCTTTTCTTTTTCTTTTTTATTTTGAGGTAGGGTATTGCTGTGTC
ACCCAGGCTGGAGTGCAGTGGTGTGATCTCGGCTCACTGCAACCTCCACCTCCCGGGCTCAGGTGATC

CTCCTGCTICAGCCTCCCAGTAACTGGGACTACAGGCGCGTGCCACATGCCIGGCTAATTTIGTATTI
TTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTCTAACTCCTGACCTCAGGTGATACGCC
CACCIGGGCCTCCCAAAATACTGGGATTACAGGCATGAGCCGCTGCATCAGCCAGCAGTT TT TCTIGT
GGTTTTTTTTGTTTGTTTTGTTTTGTTTTGTTTTTGAGATAGGGTCTTACTCTGTTGTCCACGCTGGA
GTGCTGIGGTATGATCGTAGCTCACTGCAGCCTCAAACTCCTGGGCTCAAGTGATTCCTICTGCCTCC
GCCTCCCGAGTAGCTGGGACTACAGGTATGCACCACCATACCIGGCAAAT TT TTACAAAGTT TITTGT
AGGGACGGGGICTTGCTACATTCCCCATGICGGICTTGAACTCCTGGCCTCAAGCAACTCTCCTGICT
CAGCCTCCCAAAGCACTGGGAT TACAAGTGTGAGCCACCACACCATGCCAGT TT TTCCTGITCAGTGT
GATATT TTATCT TGTTAGACTACAGTGIGT TAAAACTTGT TT TACTAAAT TT TCAAACATACTCAAAA
GIGGAGAGAATAGTATAATGAATACCCGTATGITCATCACCCATGTTTAGAATATTATTAAATATAAA
GATT TTGCTGCGTT TGICTTAGCTCT TTAAAATITT TCTT TT TCTCTT TGTGACCTAAAGGAAATTCC
ATATCT TATCACTT TACT TCTACATTCT TGACTAAGATGACTAAGACATATAGT TACATGGT TTITTG
TT =GT TT =GT TT TT TAAAGACGAAATCTCGCTCT TGICCCCCAGGCTGGAGTGCAATGGIGCCATC
TCAGCTCAGTGCAACCICTGCCTICTGGGTACAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGG
GATTACAGGCTCCTGCCACCACGCCIGGCTAATTITTGTATTITTAGTAGAGACGGCGGGGGGAGGIT
TCACCATGTTGACAAGGCTGGICTGGAACTCCTGACCTCAGGTGATCCACCCGCCTCGGCCTCCCAAA
GTGCTGGGAT TACAGGCGTGAGCCACCGCGCCCAGCCTGT TT TT TTGT TTGTGTGT TT TGTT TT ITT T

GAGACAGAGICTTGCTCTGTITCCCAGGCTGGAGTGAAGTGGIGCCATCTCAGCTCAGAGACAGAGTC
TTGCTCTGITTCCCAGGCTGGAGTGAAGIGGIGCCATCTIGGCTCACTGCAACCTICACCTCCCAGGT
TCAAGTGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCATGIGICACCACACCCGGCT
AATT TT TT TGTATT TT TAGTAGAGACGGGATT TCACCGTGTTGCCCAGGCTGGICTCGAACTCCTGAG
CTCAGGCAGICTGCCTGCCTCAGCCTCCCAAAGTGCTGGGATTACACGTGTGAACCAACCCGCCCGGC
CTGT TGIT =CT TACATAAT TCAT TATCATACCTACAAAGTTAACAGT TACTAATATCATCT TACACC
TAAATT TCTCTGATAGACTAAGGT TATT TT TTAACATCTTAATCCAATCAAATGTT TGTATCCTGTAA
TGCTCTCATTGAAACAGCTATATT TCTT TT TCAGAT TAGTGATGATGAACCAGGITATGACCITGAT T
TATTTIGCATACCTAATCATTATGCTGAGGATTIGGAAAGGGIGITTATTCCTCATGGACTAATTATG
GACAGGTAAGTAAGATCT TAAAATGAGGITTI TTACTT TT TCTIGIGT TAAT TTCAAACATCAGCAGC
TGTICTGAGTACTTGCTATTTGAACATAAACTAGGCCAACTTATTAAATAACTGATGCTTICTAAAAT
CT TCTT TATTAAAAATAAAAGAGGAGGGCCTTACTAAT TACT TAGTATCAGT TGIGGTATAGIGGGAC
TCTGTAGGGACCAGAACAAAGTAAACATTGAAGGGAGATGGAAGAAGGAACTCTAGCCAGAGICTTGC
AT TICTCAGTCCTAAACAGGGTAATGGACTGGGGCTGAATCACATGAAGGCAAGGICAGATT TT TAT T
AT TATGCACATCTAGCTTGAAAAT TT TCTGTTAAGICAAT TACAGTGAAAAACCITACCIGGTATTGA
ATGCTTGCATTGTATGICTGGCTATTCTGIGTTITTATITTAAAATTATAATATCAAAATATTTGIGT
TATAAAATAT TCTAACTATGGAGGCCATAAACAAGAAGACTAAAGT TCTCTCCT TTCAGCCT TCTGTA
CACATTICTICTCAAGCACTGGCCTATGCATGTATACTATATGCAAAAGTACATATATACATTTATAT
TT TAACGTATGAGTATAGTT TTAAATGT TATTGGACACTT TTAATATTAGIGTGICTAGAGCTATCTA
ATATAT TT TAAAGGITGCATAGCATTCTGICT TATGGAGATACCATAACTGATT TAACCAGTCCACTA
TTGATAGACACTAT TT TGTICT TACCGACTGTACTAGAAGAAACAT TCTT TTACATGT TIGGTACTIG
TICAGCTITATTCAAGTGGAATTICTGGGICAAGGGGAAAGAGTTTATTGAATATTITGGTATTGCCA
AATT TTCCICTAAGAAGT TGAATCAT TT TATACTCCTGATGT TATATGAGAGTACCTT TCTCTTCACA
ATTTGICTCTITTITTITTITTITTGAGACAAGGICTCTGTTGCCCAGGCTGGGGIGCAGTGCAGCAG
AATGATCACAGTICACTGCAGICTCAACCTCCTGGGITCAAGCGATCCTICCACCTCAGCCTCCTGAG
TAGCTGGGACTATAGGIGTGCGCCACCACTCCCAGCTAATAT TT TTAT TT TGTAGAAACAGGGT TCGC

CATGTTACCCAGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACTGGCCCAGTTTCTACAGTCTC
TCTTAATATTGTATATTATCCAGAAAATTICATTTAATCAGAACCTGCCAGICTGATAGGTGAAAATG
GTATCT TGTT TT TATT TGCATT TA
TATGATAGTGGTAT GCTT GGTT TT TT TGAAGGTATC
AAATTTTTTACCTTAT GAAACATGAGGGCAAAGGAT GT GATACGTGGAAGATTT
TTTTTA
ATGCATTTTTTTGAGACAAGGTCTTGCTCTATTGTCCAGGCTGGAGTGCAGTGGCACAATCACAGTTC
ACTCCAGCCTCAACATCCTGCACTAAAGTGATTTTCCCACCTCACCTCTCAAGTAGCTGGGACTACAG
GTACAT GCTACCAT GCCT GGCTAATT TT TT TT TT TT TGCAGGCATGGGGTCTCACTATAT TGCCCAGG

TTGGTGTGGAAGTTTAATGACTAAGAGGTGTTTGTTATAAAGTTTAATGTATGAAACTTTCTATTAAA
TTCCTGAT TT TATT TCTGTAGGACTGAACGTCTT GCTCGAGATGTGAT GAAGGAGATGGGAGGCCATC
ACATTGTAGCCCTCTGTGTGCTCAAGGGGGGCTATAAATTCTTTGCTGACCTGCTGGATTACATCAAA
GCACTGAATAGAAATAGTGATAGATCCATTCCTATGACTGTAGATTTTATCAGACTGAAGAGCTATTG
TGTGAGTATATT TAATATAT GAIT CT TT TTAGTGGCAACAGTAGGT TT TCTTATAT TT TCTT TGAATC

TCTGCAAACCATACTT GCTT TCAT TTCACT TGGT TACAGT GAGATT TT TCTAACATAT TCACTAGTAC
TT TACATCAAAGCCAATACT GT TT TT TTAAAACTAGTCACCT TGGAGGATATATACTTAT TT TACAGG
TGTGTGTGGTTTTTTAAATAAACTCCTTTTAGGAATTGCTGTTGGGACTTGGGATACTTTTTTCACTA
TACATACT GGTGACAGATACCCTCTCTT GAGCTACATCGGTT TGTGGGGAGTCAAAAGTCCT TT GGAG
CTAGGT TT GACAAATAAGGT GGGT TAACACTT GT TTCCTAGAAAGCACAT GGAGAGCTAGAGTATTGG
CGAATT GAAGAAATCCCCCT TT TT TT TTAACACACT TAAGAAAGGGGACT GCAGGTATACTCAAGAGA
GTAAGTCGCACCAGAAACCACT TT TGATCCACAGTCTGCCTGTGTCACACAATT GAAATGCATCACAA
CATT GACACT GT GGAT GAAACAAAATCAGT GT GAAT TT TAGTAGTGAATT TCAT TCATAATT
TGATCG
TGCAAACGTT TGAT TT TTAT TACT TTAGACTATT GT TI CT GATT TTAT GT TGGGTT GGTATT
TCCTGT
GAGT TACT GT TT TACCTT TAAAATAGGAAT TT TTCATACTCT TCAAAGAT TAGAACAAAT GTCCAGT
T
TTTGCTGTTTCATGAATGAGTCCTGTCCATCTTTGTAGAAACTCGCCTTATGTTCACATTTTTATTGA
GAATAAGACCACTTATCTACATTTAACTATCAACCTCATCCTCTCCATTAATCATCTATTTTAGTGAC
CCAAGT TT TT GACCTT TTCCAT GT TTACATCAATCCTGTAGGTGAT TGGGCAGCCATT TAAGTATTAT
TATAGACATTTTCACTATCCCATTAAAACCCTTTATGCCCATACATCATAACACTACTTCCTACCCAT
AAGCTCCTTTTAACTTGTTAAAGTCTTGCTTGAATTAAAGACTTGTTTACGGTATCGATAAGCTTGAT
ATCAAAACGCCAACTTTGACCCGGAACGCGGAAAACACCTGAGAAAAACACCTGGGCGAGTCTCCACG
TAAACGGTCAAAGTCCCCGCGGCCCTAGACAAATATTACGCGCTATGAGTAACACAAAATTATTCAGA
TTTCACTTCCTCTTATTCAGTTTTCCCGCGAAAATGGCCAAATCTTACTCGGTTACGCCCAAATTTAC
TACAACATCCGCCTAAAACCGCGCGAAAATTGTCACTTCCTGTGTACACCGGCGCACACCAAAAACGT
CACTTTTGCCACATCCGTCGCTTACATGTGTTCCGCCACACTTGCAACATCACACTTCCGCCACACTA
-CTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATATTG
GC TTCAATCCAAAATAAGGTATAT TATTGATGATGT TTAAACAT TAAGAATTAATT CGAT CC T GAAT G
GCGAAT GGACGCGCCCTGTAGCGGCGCATTAAGCGCGCGGGT GT GGTGGT TACGCGCAGCGT GACCGC
TACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCG
GCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGAGCTTTACGGCACCTC
GACCGCAAAAAACTTGATTTGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCG
CCCT TT GACGTT GGAGTCCACGTTCT TTAATAGT GGACTCTT GT TCCAAACT GGAACAACACTCAACC
CTATCGCGGTCTATTCTTTTGATTTATAAGGGATGTTGCCGATTTCGGCCTATTGGTTAAAAAATGAG
CT GATT TAACAAAAAT TT TAACAAAATTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCT
GCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCA
GCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGAT

GAATCCAGAAAAGCGGCCATTITCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGICACGACGA
GATCCICGCCGICGGGCATGCTCGCCITGAGCCIGGCGAACAGITCGGCTGGCGCGAGCCCCTGATGC
TCTICGICCAGATCATCCTGATCGACAAGACCGGCTICCATCCGAGTACGTGCTCGCTCGATGCGATG
TITCGCTIGGIGGICGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCA
TGAIGGATACTITCTCGGCAGGAGCAAGGIGAGATGACAGGAGATCCIGCCCCGGCACTICGCCCAAT
AGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGC
CAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAA
AAAGAACCGGGCGCCCCIGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGICTGITGT
GCCCAGICATAGCCGAATAGCCICTCCACCCAAGCGGCCGGAGAACCIGCGTGCAATCCATCTIGTIC
AATCATGCGAAACGATCCICATCCIGICICTIGATCAGAGCTIGATCCCCIGCGCCATCAGATCCTIG
GCGGCAAGAAAGCCATCCAGITTACTITGCAGGGCTICCCAACCITACCAGAGGGCGCCCCAGCTGGC
AATTCCGGITCGCTIGCTGICCATAAAACCGCCCAGICTAGCTATCGCCATGTAAGCCCACTGCAAGC
TACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCCGGG
GICAGCACCGTTICTGCGGACTGGCTTICTACGTGAAAAGGATCTAGGIGAAGATCCITITTGATAAT
CICATGGCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTIC
CCGGCAACAATTAATAGACTGGAIGGAGGCGGATAAAGITGCAGGACCACTICTGCGCTCGGCCCTIC
CGGCTGGCTGGITTATTGCTGATAAATCTGGAGCCGGIGAGCGIGGGICTCGCGGIATCATTGCAGCA
CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGA
TGAACGAAATAGACAGATCGCTGAGATAGGIGCCICACTGATTAAGCATIGGTAACTGICAGACCAAG
ITTACTCATATATACTITAGATTGATTTAAAACTICATTITTAATTTAAAAGGATCTAGGIGAAGATC
CTTITTGATAATCTCATGACCAAAATCCCITAACGTGAGITTICGTICCACTGAGCGICAGAC
RightITR = first underlined and bold sequence CBh = first underlined sequence mCherry:PKD1 = first bold sequence HGHpA = second underlined sequence EFla = second bold sequence PKD2 = third underlined sequence BGHpA = third bold sequence Packaging Signal = fourth underlined sequence LeftITR = second underlined and bold sequence SEQ ID NO:8 HDAd- SAM
Ri ghtITR-U6VM-CMV-dC as9VP 64-HGHpA-EF 1 a-MPH-HGHpA-P ackagi ng Si gnal -L eftITR
CCCGTAGAAAAGATCAAAGGATCTICTIGAGATCCITITITICTGCGCGTAATCTGCTGCTIGCAAAC
AAAAAAACCACCGCTACCAGCGGIGGITTGITTGCCGGATCAAGAGCTACCAACTCTITTICCGAAGG
TAACTGGCTICAGCAGAGCGCAGATACCAAATACTGICCTICTAGIGTAGCCGTAGITAGGCCACCAC

TTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG
TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGG
GCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTA
CAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG
CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTG
TCGGGT TTCGCCACCTCTGACT TGAGCGTCGATT TT TGTGATGCTCGTCAGGGGGGCGGAGCCTATGG
AAAAACGCCAGCAACGCGGCCT TT TTACGGITCCIGGCCT TT TGCTGGCCTT TTGCTCACATGT TCTT
TCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCC
GCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTT
CTCCTTACGCATCTGTGCGGTATTTCACACCGCATATGGATCCATGCATGTTAAGT T TAAACATCATC
AATAATATAC C T TATT TTGGAT TGAAGC CAATATGATAATGAGGGGGTGGAG TT TG TGAC GTGGCGC
G
GGGCGTGGGAACGGGGCGGGTGACGTAGGT TT TAGGGCGGAGTAACTTGTATGTGT TGGGAATTGTAG
TT TI CT TAAAATGGGAAGTTACGTAACGTGGGAAAACGGAAGTGACGATT TGAGGAAGTTGIGGGIT T
TT TGGCTT TCGT TTCTGGGCGTAGGT TCGCGTGCGGTT TTCTGGGTGT TT TT TGTGGACT TTAACCGT
TACGTCATTTTTTAGTCCTATATATACTCGCTCTGCACTTGGCCCTTTTTTACACTGTGACTGATTGA
GCTGGTGCCGTGTCGAGTGGTGTTTTTTGATGCCCCCCCTCGAGGTTCGACGGTATCGATAAGCTTGA
TT TAAT TAAGGCCGGCCCCTAGGGGCGCGCGCGGCCGCTAGGGATAACAGGGTAATGAGGGCCTATT T
CCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGAC
TGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAG
TT TTAAAATTATGT TT TAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTAT TTCGAT TTCTTG
GC T T TATATATCTT GT GGAAAGGACGAAACACCGOARIIOOMARMNWOAOMNGT TT TAGAGCTAGG
CCAACATGAGGATCACCCATGICTGCAGGGCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT
GGCCAACATGAGGATCACCCATGTCTGCAGGGCCAAGTGGCACCGAGTCGGTGCTTTTTTTGGATCCT
GI TGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCAT
GGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGA
CCGACCGGCTCGGGTTCTCCCGGGACTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTG
ACCCTGTTCATCAGCGCGGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCG
CGGCCTGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGC
CGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAAC
TGCGTGCACTTCGTGGCCGAGGAGCAGGACTGATAGGGATAACAGGGTAATGCTAGCATAGTAATCAA
TTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCG
CC TGGC TGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGTC
AATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAAC TGCC CAC T TGGCAG TACATC
AAGTGTATCATATGCCAAGTAC GC CC CC TATTGACG TCAATGAC GG TAAATGGC CC GC C
TGGCATTAT
GC CCAG TACATGAC C T TATGGGAC TT TC C TAC TTGGCAGTACATC TAC GTAT TAGTCATC GC
TATTAC
CATGGTGATGCGGT TT TGGCAG TACATCAATGGGCG TGGATAGC GG TT TGAC TCACGGGGATTTCCAA
GTC TCCAC CC CATTGACG TCAATGGGAG TT TG TT TTGCAC CAAAATCAAC GGGAC T
TTCCAAAATGTC
GTAACAAC TCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTC TATATAAGCAGA
GC TC GT TTAG TGAACC GTCAGATC GC C TGGAGAC GC CATC CACGC TGT TT TGAC C
TCCATAGAAGACA
CCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGCCGGGAACGGTGCATTGGAACGCGGATTCCC
CG TGCCAAGAGTGACG TAAG TACC GC C TATAGAG TC TATAGGCCCACAAAAAATGC TT TC
TTCTTTTA
ATATAC TT TT TTGT TTATC T TATT TC TAATAC TT TCCC
TAATCTCTTTCTTTCAGGGCAATAATGATA
CAATGTATCATGCC TC TT TGCACCAT TC TAAAGAATAACAGTGATAATTTCTGGGTTAAGGCAATAGC

AATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTGATGTAAGAGGTTTCATATTGCTAAT
AGCAGCTACAATCCAGCTACCATTCTGCTTTTATTTTATGGTTGGGATAAGGCTGGATTATTCTGAGT
CCAAGCTAGGCCCTTTTGCTAATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGT
GCTGGTCTGTGTGCTGGCCCATCACTTTGGCAAAGAATTGGGATCGTACGGCCACCATGAAAAGGCCG
GC GGCCAC GAAAAAGGCC GGCCAGGCAAAAAAGAAAAAGGACAAGAAG TACAGCAT CGGC C T GGCCAT
CGGCACCAAC T C T GT GGGCT GGGCCGT GAT CACCGACGAGTACAAGGT GCCCAGCAAGAAAT TCAAGG

T GCT GGGCAACACCGACCGGCACAGCAT CAAGAAGAACCT GAT CGGAGCCCT GC T GT T CGACAGCGGC

GAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGAT
CT GC TAT C T GCAAGAGAT CT T CAGCAACGAGAT GGCCAAGGT GGACGACAGC T T CT
TCCACAGACTGG
AAGAGT CCIT CC T GGT GGAAGAGGATAAGAAGCACGAGCGGCACCCCAT C T T CGGCAACAT CGT
GGAC
GAGG T GGC C T AC CACGAGAAGT AC CC CACCAT C T AC CACC T GAGAAAGAAAC T GGT
GGACAGCACCGA
CAAGGCCGACCT GCGGCT GAT C TAT C T GGCCCTGGCCCACAT GAT CAAGT T CCGGGGCCACT TCCT
GA
T CGAGGGCGACC T GAACCCCGACAACAGCGACGT GGACAAGC T GT T CAT CCAGC T GGT
GCAGACCTAC
AACCAGCT GT T CGAGGAAAACCCCAT CAACGCCAGCGGCGT GGACGCCAAGGCCAT CC T GTC T GCCAG

AC T GAGCAAGAGCAGACGGC T GGAAAAT CT GAT CGCCCAGCT GCCCGGCGAGAAGAAGAAT GGCCT GT

T CGGCAACCT GAT T GCCC T GAGCCTGGGCC T GACCCCCAACT TCAAGAGCAACT TCGACC T
GGCCGAG
GAT GCCAAAC T GCAGC T GAGCAAGGACACC TACGACGACGACCT GGACAACC T GCT GGCCCAGAT
CGG
CGACCAGTACGCCGACCT GT T T CT GGCCGCCAAGAACC T GTCCGACGCCAT CCT GC T GAGCGACAT
CC
T GAGAGT GAACACCGAGAT CAC CAAGGCCCCCCT GAGC GCCT CTAT GAT CAAGAGATACGAC GAGCAC

CACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTT T
CT TCGACCAGAGCAAGAACGGCTACGCCGGCTACAT TGACGGCGGAGCCAGCCAGGAAGAGT TCTACA
AGT T CAT CAAGC CCAT CC T GGAAAAGAT GGAC GGCACC GAGGAACT GC T C GT GAAGCT
GAACAGAGAG
GACCTGCTGCGGAAGCAGCGGACCITCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCT
GCACGCCAT T CT GCGGCGGCAGGAAGAT TT TTACCCAT TCCTGAAGGACAACCGGGAAAAGATCGAGA
AGAT CC T GACCT TCCGCAT CCCCTAC TACGT GGGCCCT CT GGCCAGGGGAAACAGCAGAT TCGCCTGG

AT GACCAGAAAGAGCGAGGAAACCAT CACCCCCT GGAACT TCGAGGAAGIGGIGGACAAGGGCGCTIC
CGCCCAGAGC T T CAT CGAGCGGAT GACCAACT T CGATAAGAACC T GCCCAACGAGAAGGT GC T
GCCCA
AGCACAGCCT GC T GTACGAGTACT TCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAG
GGAAT GAGAAAGCCCGCC TT CC T GAGCGGCGAGCAGAAAAAGGCCAT CGT GGACCT GC T GIT
CAAGAC
CAACCGGAAAGT GACCGT GAAGCAGC T GAAAGAGGACTAC T T CAAGAAAAT CGAGT GC T T CGAC T
CCG
T GGAAAT C T CCGGCGT GGAAGAT CGGT T CAACGCCT CCCT GGGCACATACCACGAT CT GC T
GAAAAT T
AT CAAGGACAAGGACT TCCTGGACAATGAGGAAAACGAGGACAT T C T GGAAGAT AT CGT GCT GACCC
T
GACACT GT T T GAGGACAGAGAGAT GAT CGAGGAACGGC T GAAAACC TAT GCCCACCTGT T CGAC
GACA
AAGT GAT GAAGCAGCT GAAGCGGCGGAGATACACCGGC T GGGGCAGGC T GAGCCGGAAGC T GAT CAAC

GGCAT CCGGGACAAGCAGT CCGGCAAGACAAT CC T GGAT T TCCTGAAGTCCGACGGCT TCGCCAACAG
AAAC TT CAT GCAGC T GAT CCAC GACGACAGCC T GACCT T TAAAGAGGACAT
CCAGAAAGCCCAGGTGT
CCGGCCAGGGCGATAGCC T GCACGAGCACAT T GCCAAT CT GGCCGGCAGCCCCGCCAT TAAGAAGGGC
AT CC T GCAGACAGT GAAGGT GGT GGACGAGCT CGT GAAAGT GAT GGGCCGGCACAAGCCCGAGAACAT

CG T GAT CGAAAT GGCCAGAGAGAACCAGAC CACC CAGAAGGGACAGAAGAACAGCC GC GAGAGAAT GA
AGCGGAT C GAAGAGGGCAT CAAAGAGC T GGGCAGCCAGAT CC T GAAAGAACACC CC GT GGAAAACAC
C
CAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGA
AC T GGACAT CAACCGGCT GT CCGACTACGAT GTGGACCACAT CGT GCC T CAGAGCT TT CT
GAAGGACG
AC T CCAT CGACAACAAGGT GCT GACCAGAAGCGACAAGGCCCGGGGCAAGAGCGACAACGT GCCCT CC

GAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAG
AAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCA
AGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAAC
ACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGT
GTCCGATT TCCGGAAGGATT TCCAGT TT TACAAAGT GCGCGAGATCAACAACTACCACCACGCCCACG
ACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTC
GT GTACGGCGACTACAAGGT GTACGACGTGCGGAAGAT GATCGCCAAGAGCGAGCAGGAAATCGGCAA
GGCTACCGCCAAGTACTTCT TCTACAGCAACATCAT GAACTT TT TCAAGACCGAGATTACCCTGGCCA
ACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGT GT GGGATAAG
GGCCGGGATT TT GCCACCGT GCGGAAAGTGCT GAGCAT GCCCCAAGTGAATATCGT GAAAAAGACCGA
GGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCA
GAAAGAAGGACT GGGACCCTAAGAAGTACGGCGGCT TCGACAGCCCCACCGT GGCCTATTCT GT GCT G
GT GGTGGCCAAAGT GGAAAAGGGCAAGTCCAAGAAACT GAAGAGTGTGAAAGAGCT GCTGGGGATCAC
CATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAG
TGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCT GT TCGAGCTGGAAAACGGCCGGAAGAGA
AT GCTGGCCTCT GCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATAT GT GAACT T
CCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGT
TTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTG
ATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAG
AGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGT
ACTT TGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCT GGACGCCACCCT GATC
CACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCGACAGCGCTGG
AGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTAGCGGACCTAAGAAAAAGAGGAAGGTGG
CGGCCGCTGGATCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCC
CTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGG
CAGT GACGCCCT TGAT GATT TCGACCTGGACATGCT GATTAACT GT ACAT AAACGGGTGGCATCCC TG
TGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAA
TAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTG
GTATGGAGCAAGGGGCAAGT TGGGAAGACAAC C TGTAGGGCC TGCGGGGTC TAT TGGGAACCAAGC TG
GAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTC
AGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAG
AGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTG
GCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTGAATT CT AACT AT
AACGGTCCTAAGGTAGCGAAGCTAGCTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCA
GCTAATGGACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGC
ACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTG
GCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAAC
CGTATATAAGTGCAGTAGTCGCCGTGAACGTTCT TT TTCGCAACGGGT TT GCCGCCAGAACACAGGTA
AGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTAC
TTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGA
GGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGC
CGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAA
TT TT TGAT GACCTGCT GCGACGCT TT TT TI CT GGCAAGATAGTCTT GTAAAT
GCGGGCCAAGATCTGC

ACACTGGTAT TTCGGT TT TTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCG
GCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGICTCAAGCTGGCCGGCCTGC
TCTGGIGCCIGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGICGGCAC
CAGTTGCGTGAGCGGAAAGATGGCCGCTICCCGGCCCTGCTGCAGGGAGCTCAAAATGAAGGACGCGG
CGCTCGGGAGAGCGGGCGGGTGAGICACCCACACAAAGGAAAAGGGCCITTCCGTCCTCAGCCGTCGC
TTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTA
CGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACT
GAAGTTAGGCCAGCTIGGCACT TGATGTAATTCTCCTIGGAATT TGCCCT TT TTGAGT TIGGATCTIG
GT TCAT TCTCAAGCCTCAGACAGIGGITCAAAGT TT TT TI CT TCCATT TCAGGIGTCGTGACGTACGG
CCACCATGGCTTCAAACTTTACTCAGTTCGTGCTCGTGGACAATGGTGGGACAGGGGATGTGACAGTG
GC TC C T TC TAAT TTCGC TAATGGGGTGGCAGAGTGGATCAGC TC CAAC TCAC GGAGCCAGGC C
TACAA
GGTGACATGCAGCGTCAGGCAGTCTAGTGCCCAGAAGAGAAAGTATACCATCAAGGTGGAGGTCCCCA
AAGTGGCTACCCAGACAGTGGGCGGAGTCGAACTGCCTGTCGCCGCTTGGAGGTCCTACCTGAACATG
GAGCTCACTATCCCAATTTTCGCTACCAATTCTGACTGTGAACTCATCGTGAAGGCAATGCAGGGGCT
CC TCAAAGAC GG TAATCC TATC CC TTCC GC CATC GC CGC TAAC TCAGG TATC TACAGC GC
TGGAGGAG
GTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTAGCGGACCTAAGAAAAAGAGGAAGGTGGCGGCC
GC TGGATCCCC T TCAGGGCAGATCAGCAACCAGGCCC TGGC TC TGGCCCC TAGC TCCGC TCCAGTGC T

GGCCCAGACTATGGTGCCCTCTAGTGCTATGGTGCCTCTGGCCCAGCCACCTGCTCCAGCCCCTGTGC
TGAC CC CAGGAC CACC CCAG TCAC TGAGCGC TCCAG TGCC CAAG TC TACACAGGCC
GGCGAGGGGAC T
CTGAGTGAAGCTCTGCTGCACCTGCAGTTCGACGCTGATGAGGACCTGGGAGCTCTGCTGGGGAACAG
CACC GATC CC GGAG TG TTCACAGATC TGGC C TCC GTGGACAAC TC TGAGT TTCAGCAGC TGC
TGAATC
AGGGCG TG TC CATG TC TCATAG TACAGC CGAACCAATGC TGATGGAGTAC CC CGAAGC CATTAC
CCGG
CTGGTGACCGGCAGCCAGCGGCCCCCCGACCCCGCTCCAACTCCCCTGGGAACCAGCGGCCTGCCTAA
TGGGC TGTCC GGAGATGAAGAC TTC TCAAGCATC GC TGATATGGAC TT TAGTGC CC TGC
TGTCACAGA
TT TCC TC TAGTGGGCAGGGAGGAGGTGGAAGCGGC T TCAGCGTGGACACCAGTGCCC TGC TGGACC TG
TTCAGCCCCTCGGTGACCGTGCCCGACATGAGCCTGCCTGACCTTGACAGCAGCCTGGCCAGTATCCA
AGAGC TCC TG TC TC CC CAGGAGCC CC CCAGGC C TCC CGAGGCAGAGAACAGCAGCC
CGGATTCAGGGA
AGCAGC TGGTGCAC TACACAGC GCAGCC GC TG TTCC TGC TGGAC CC CGGC TC CG TGGACACC
GGGAGC
AACGACCTGCCGGTGCTGTTTGAGCTGGGAGAGGGCTCCTACTTCTCCGAAGGGGACGGCTTCGCCGA
GGACCCCACCATCTCCCTGCTGACAGGCTCGGAGCCTCCCAAAGCCAAGGACCCCACTGTCTCCTGTA
CATAAACGGGIGGCATCCCIGTGACCCCTCCCCAGTGCCICTCCTGGCCCTGGAAGTTGCCACTCCAG
TGCCCACCAGCCTIGTCCTAATAAAATTAAGTTGCATCATTITGICTGACTAGGIGTCCTICTATAAT
AT TATGGGGIGGAGGGGGGIGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCIGTAGGGCCTGCGG
GGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGG
TTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGC
TAATTITTGITTITTIGGTAGAGACGGGGITTCACCATATTGGCCAGGCTGGICTCCAACTCCTAATC
TCAGGTGATCTACCCACCTIGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTICC
CTGICCITGAATTCTAACTATAACGGICCTAAGGTAGCGAAGTCGACCGAATCGTTGICCCITGICAC
AGCCAT TGAGAATT TTGGCAGGGAGCATGT TCTTAGAGCATT TT TAGGCTCTGCGGGACATAACAGCT
CTGCCTCAGAGCACATGCCT TTCTCAGCTCCTGAAAGCCACTGATCAAAT TGGAACAT TT TGTACCT T
AGGGATGAGGATATCAACTCTCCCAGCCACTTAGAGGGATAAATGTGATGATGCATTCAATTGTGACT
ACATCTGATCCCAACTGTTGCTICAGCTGCTCTCCTATAGCACATGGCGGGAGGCGTGCATCCCAGTA
GCTACCTCCCCACTITTGGGGAGATGIGGITCCATCCATGAAACCIGGGTACCCGCCTACCAGGICCT

GGCCTATCAGGT GGCAGGGTCT GGTCAAAGAAGGGCAT GT GT GGTCTTCAGCAAGGGAGACAGGACGG
TGGTGCAGAGCGTCTAGACCCTCAGGGCAAGTCTCCCCCACACCTGCTCCCGGGGCAGTTGTCTTTGT
GACCTCCCATCCCCCTCTGTTTCATCCTCTATAAAATGAGGGGCTGAGCCCCAAAATAACAGGCTTCT
TT GCCATGAT GCAAAACT GCTGAATCTT TCTT TCTGACACACAAGGCATCGAGCAGCCTCTGAAAGAA
CCAAAGCCACTAGCAGGCTTCCTGACTT GGGT TT GTAGGTACTGAATACTCCCT TGAAAAATAAAAAC
ATAGAGGCACTTTTCTCCTGGCTGTTTATTACAGAACGAAGAAAAAACACACTGGCTTGAAACAGACG
CCAGAT TT CAAAT GTAGAGGT GAAATACGAGGT GGCAAT TAAAAT GT GAT TACAGAAAGT CT
GGACAC
TGAGAAAAGT TTACAGGACAGT GGGT GT GGGT TT TCTATAACAGACACTTAAATATACAT GACGATAA
TTGCAGATAGAAACCATCAAAGACAAACCCCAAATCAACTAATAATGTTTACAGATGTTCCCCCCCAA
ACCACAGAGCCTTACATCAAAACAAATACTGAAAGGCTTTAAACCAGGAACAGCTCGCCTTAACCCCA
CGAGGGTGCACACAAGCTGGGCTTTTTCTCTCGGTCTGAATGGTAAAGGGAGGAGGATACTCTAGCTC
CTCCAGGTGGATTGCTGAGACAGGGCTCGGCTCACACACTGTCTCTGCGCCTCTCCCAAATCTGGAGA
ACTCTCCCAGCCTCCTGGTAAAGTGTCTCTGTGGGGCACTTAACGATAAAACAGCTTCTGCTGTAAAG
CT CAT TAGGAAAGAGC TAGC GGAGAC T GAAAGGT TCGCAAAAGAGATTAAGAAT CACACAAGGCAATA
GGAT TT TTAGTGAACATAGAAATAAATGGCCAAGTGGT TT TCTATT TGGCAT TT GTCAACTT GCACAA
CAACTCTTGGTCATATCCACATTGCTCATTGCATTAAAACCATAAGCGACTCAGCCACCTAGCTTAAC
AAGGTATCACTGGAGCAAACAACACGGTCT GCATAT TT GTAACATT GTATAATAAACACAAAACAAT G
CATAGTAAACACAACTCTACTGAAACAAAAGCCGTCGCTTTATTTACAAAGTCACAAAATGAAGTATA
AATACT TCTGTCAT TAAT GT TTAGGAAAACCATT TACAAAAT TT TCAAATAT GTACACGTAGCT TGAA
AAATCACCAGCT TTCCAT TT TGTCACAGGTAGAGAGAGGGATAAGCAT GGGCTGACAACACCACTCAA
AT TGTAACGGGAGACAACTGCGGGTATGGATCGACACCACTTCCTAGAGT GATGTCACCATGGGGGT T
TCTATGGGCATCCTGCTCAGATTTAAAGTGCCCCAGCATCCTGGGTGACTTGCCCAGAATTCTGGGCT
GT GGCATT TT GAGCAGCAGCAT GCTGTTCCAAAATGTCGTCGATCAGCCTCAAGTT GCACACCCAGTC
TTCATCTGGGCTCACACAGGAGCCTTTCAAGAGAGCTTCAATGAAATCTACCTCATTGCAGTCAGGTG
ACGAAATCAGATCATTTAGTGGGGGTTGGGGCTGGCGCAAAAAGTCGGCAGGTGGCAGCTCAGGGGGA
ATATCCGTTCTGTCGAACGGACCTGGGAACTGGCTGGCAGCAACGGCAGAAGCAGCAGCAGCGGTGGC
AGCAGCAGCCACATAGCTTGGTGGCTCGATGCCCTGTATGGGGCTCAGGGGACTAAAGCTGGCCATAC
CCTGCTGGAGGAACTTGGTGGTGTTTGCTACAGGCACCGGGCCCTGTACCGGGCTCTGCCTGAGGCTC
TGGCTGCCCAGCAGGCTGAAGCTGGGGTTGTTGGCCAGGGGCACTTGTGTTCCCATCGCAGCGGGCAC
TTGTGCCTCCCAATCAGATGGCCTCTGAAGGCAGGCCTGGCCAGAAGGTGAGTGCTGCTGAACGCTAT
TATCCACTTGGCTGAGGGGTGTTTTCCCCGAAACTGCTGTGGTCACAGCTGCTGCCGCTGTGACCCAT
GCAGCATT GT TGAACGCAGT GGGCAT TCTT GGCACACTAGGCCGTCTGAGCT GGTGGGGACTCAAGGA
CTGGGTGCCCAGGGAGCTGGGACAGAACCCAGGCAGGGGCACTTCTGGTGGGGTGGCCTTGGGGCTCT
GCATATGCTGGCAGACAGAGTCAAGTCTGCCCAGGGGAGTCTGGCCTGAGTGTGAGAGGATGGGACAC
TGGGGGCTGGAGGTGAAAATTCCTTGCCGCTTCCCCAGAGTTGGTGAGATCACTCCCATGCCCTCGCA
GCTCTGGTGCCTGGTGAGTGGGATCATTCCTGGACTCAGATTGTTCTGAAGAAGCCCAGTTCTGGGTG
GCATCAAGTGCTTGCTAGATGGGGGGCTTGCCTTGATCCGGCTACACTTGGAGGTGACTTGTTCTTGG
ACGGCTACATACAGAAAGAGAGAAGTGGGGATGAGTTCCAAAGGCATCCTCGACTTCGGCTGTGGCCA
CCGGAGGGTAGCTCCTGGCCCAACACGGACTTCTCACCTCCCGCCCTTGGCTCTCTACTGAGCTCCCC
CCTGCTCCCCAATTCCTCGCCATTCCCCTCATTTCTCTGCCCTCAGCCTGGACTGCAGTTCTTCTGGG
AAGCTGCCCCAACTCCCTAGGTCT GT GCTCACCAAGAGCAGATCACACTGGACT GAAATGCCAGCTGA
TTTGTCTCTTCAAGAAAATTGGAAGCTCCTGGAGGTCAGGGTCCATGTCTGCTTTTACACTCAGTGCT
CT GTAT GCAGGCCT GGCACT GCCCACCCTT TGACAGGT GGTGCATATT TT GTAGAAGGAAGGAAGGGG

CCAGGTGGGGTGGGCTGGGCTGGTGGCGGGAGCTAGCTCAGCCTCTTAGATTCTCTACCCGATGGATG
TGACCTGGGACAGCAAGTGAGTGTGGTGAGTGAGTGCAGACGGTGCTTTGTTCCCCTCTTGTCTCATA
GCCTAGATGGCCTCTGAGCCCAGATCTGGGGCTCAGACAACATTTGTTCAACTGAACGGTAATGGGTT
TCCTTTCTGAAGGCTGAAATCTGGGAGCTGACATTCTGGACTCCCTGAGTTCTGAAGAGCCTGGGGAT
GGAGAGACACGGAGCAGAAGATGGAAGGTAGAGTCCCAGGTGCCTAAGATGGGGAATACATCTCCCCT
CATTGTCATGAGAGTCCACTCTAGCTGATATCTACTGTGGCCAATATCTACCGGTACT TT TT TGGGGT
GGACACTGAGTCATGCAGCAGTCTTATGGTTTACCCAAGGTCAGGTAGGGGAGACAGTGCAGTCAGAG
CACAAGCCCAGTGTGTCTGACCCACCCAAGAATCCATGCTCGTATCTACAAAAATGAT TT TT TCTCT T
GTAATGGTGCCTAGGTTCTTTTATTATCATGGCATGTGTATGTTTTTCAACTAGGTTACAATCTGGCC
TTATAAGGTTAACCTCCTGGAGGCCACCAGCCTTCCTGAAACTTGTCTGTGCTGTCCCTGCAACTGGA
GTGTGCCTGATGTGGCACTCCAGCCTGGACAAGTGGGACACAGACTCCGCTGTTATCAGGCCCAAAGA
TGTCTTCCATAAGACCAGAAGAGCAATGGTGTAGAGGTGTCATGGGCTACAATAAAGATGCTGACCTC
CTGTCTGAGGGCAAGCAGCCTCTTCTGGCCCTCAGACAAATGCTGAGTGTTCCCAAGACTACCCTCGG
CCTGGTCCAATCTCATCCCACTGGTGCGTAAGGGTTGCTGAACTCATGACTTCTTGGCTAGCCTGCAA
CCTCCACGGAGTGGGAACTACATCAGGCAT TT TGCTAACTGCTGTATCCTAGGCCAATAAATGT TGAT
CACATTTATAGCTGCCATGGTAGGGTGGGGACCCCTGCTATCTATCTGTGGAGGCTCTGGGAGCCCCT
GACACAAACTTTCTGAAGCAGAGCCTCCCCAACCCCTTTTCCATTCCCTATACCTGACAGATGGCCCA
GGAACCCATTAGAAATGGAAGGTCACTGCAGCAGTATGTGAATGTGCGTGTGGGAGAAGGGCAGGATC
AGAGCCCTGGGGGTGTGGCAGCCCCCAAGTGATTCTAATCCAGATCCTAGGGTTGTTTCCCTGTCCCA
TTGAAATAGCTGCT TTAAGGGGCCTGACTCAGGGAAATCAGTCTCT TGAATTAAGTGGTGAT TT TGGA
GTCATTTAGACCAGGCCTTCAATTGGGATCCACTAGTTCTAGAGCGGCCGGGCCCAGGGAACCCCGCA
GGCGGGGGCGGCCAGTTTCCCGGGTTCGGCTTTACGTCACGCGAGGGCGGCAGGGAGGACGGAATGGC
GGGGTTTGGGGTGGGTCCCTCCTCGGGGGAGCCCTGGGAAAAGAGGACTGCGTGTGGGAAGAGAAGGT
GGAAATGGCGTTTTGGTTGACATGTGCCGCCTGCGAGCGTGCTGCGGGGAGGGGCCGAGGGCAGATTC
GGGAATGATGGCGCGGGGTGGGGGCGTGGGGGCTTTCTCGGGAGAGGCCCTTCCCTGGAAGTTTGGGG
TGCGATGGTGAGGTTCTCGGGGCACCTCTGGAGGGGCCTCGGCACGGAAAGCGACCACCTGGGAGGGC
GTGTGGGGACCAGGTTTTGCCTTTAGTTTTGCACACACTGTAGTTCATCTTTATGGAGATGCTCATGG
CCTCATTGAAGCCCCACTACAGCTCTGGTAGCGGTAACCATGCGTATTTGACACACGAAGGAACTAGG
GAAAAGGCATTAGGTCATTTCAAGCCGAAATTCACATGTGCTAGAATCCAGATTCCATGCTGACCGAT
GCCCCAGGATATAGAAAATGAGAATCTGGTCCTTACCTTCAAGAACATTCTTAACCGTAATCAGCCTC
TGGTATCTTAGCTCCACCCTCACTGGTTTTTTCTTGTTTGTTGAACCGGCCAAGCTGCTGGCCTCCCT
CCTCAACCGTTCTGATCATGCTTGCTAAAATAGTCAAAACCCCGGCCAGTTAAATATGCTTTAGCCTG
CTTTATTATGATTATTTTTGTTGTTTTGGCAATGACCTGGTTACCTGTTGTTTCTCCCACTAAAACTT
TT TAAGGGCAGGAATCACCGCCGTAACTCTAGCACT TAGCACAGTACT TGGCTTGTAAGAGGTCCTCG
AT GAT GGT TT GT TGAATGAATACATTAAATAATTAACCACTT GAACCCTAAGAAAGAAGCGATT CTAT
TTCATATTAGGCATTGTAATGACTTAAGGTAAAGAGCAGTGCTATTAACGGAGTCTAACTGGGAATCC
AGCTTGTTTGGGCTATTTACTAGTTGTGTGGCTGTGGGCAACTTACTTCACCTCTCTGGGCTTAAGTC
ATTTTATGTATATCTGAGGTGCTGGCTACCTCTTGGAGTTATTGAGAGGATTATAAGACAGTCTATGT
GAATCAGCAACCCT TGCATGGCCCCTGGCGGGGAACAGTAATAATAGCCATCATCATGTT TACT TACA
TAGTCCTAAT TAGTCT TCAAAACAGCCCTGTAGCAATGGTATGATTAT TACCAT TT TACAGATGAGGA
ACCTTTGAAGCCTCAGAGAGGCTAACAGACATACCCTAGGTCATACAGTTATTAAGAGAAGGAGCTCT
GTCTCGAACCTAGCTCTCTCTCTCTCGAGTAATACCAGTTAAAAAATAGGCTACAAATAGGTACTCAA
AAAAATGGTAGTGGCTGT TGTT TT TATTCAGT TGCTGAGGAAAAAATGTTGATT TT TCATCTCTAAAC

ATCAACTTACTTAATTCTGCCAAT IT CT TT TT TT TGAGACAGGGICTCACTCTGICACCTAGGATGGA
GTGCAGTGGCACAATCACTGCTCACTGCAGCCTCGACT TCCCGGGCTCGGGTGATTCTCCCCAGGCTC
AGGGGATTCTCCCACTICAGCCTCCCAAGTAGCTGGGACTACAGGIGCGCACCACCATCCCTGGCTAA
TATTTGTACTTTATTTTATTTATTTATTTATTTATTTTTTGAGATGGAGTTTCGCTCTTGTTGCCCGG
GCTGGAGTACAGIGGCATGATCTCGGCTCAGTGCAACCICTGCCTCCCGGGITCAAGCGATTCTCCTA
CCTCATCCCCCTGAGTAGCTGGGATTACAGGCGCCTGCCACCATGCCIGGCTAATT TT TIGTATITT T
AATAGAGACGAGGITTCACCATGTTGGCCAGGCTACTCTCGAACTCCTGATCTCAGGTGATCCACCCG
CCTIGGCCTCCCAAAGTGCTGGGATTACAGGCGTGAGCCACTGCGCCCGGCCTAATATTIGTATITTI
TGTAGAGATGGIGTITTGCCATGTTGICCAGGCTGGICTTGAACTCCTGAGCTCAAGCGATCTGCCCG
CCICTGCTICCCAAAGTGCTGGGATTACAGGCATGAGCCACCGTGCCIGGCCTAGGTAGACGCTITTA
GCTITGGGGIGTGATGCCTGCCCCAGTATATAGTGAATTTAATTATTGCTAGAGCTGGCTGITTGTTA
GT TT TCTT TGAACATAAGATACTCAT TGIT TT TAGT TIGCAAATCCCICT TCCT TT TTAAAAAATTTC

TITCCCITAAATTGITTGCATGTTAGCAATAACAAATGCTTAAATGGIGCTATGTGCTAGATACTCTT
CTAAGCCCTGTTATGTATAT TAACTAAT TT TT TAAATTACACAAATCAGAGAGGTTAAGTAACT TGCC
CAAGATTACCCAACAATACTAGGATTTGAACCTAAGITTGICTCACCCCAGATTCTGCTCTTAATCTC
TAAACT TT TAAGTTAGTAGTGACAATAGTAGGTATT TATTGAATACTTAACTATGT TT TAGGCGTTGA
AGTAAATATT TTGCAGGCAT TATCTAATGTAAACACCCTAAAGT TACATAACAGGTACCCTT TAGGTA
AATAAACACTAGTATGACCTTGGAGGCACAGATAGTTGAAGTAACTTGCCCAATATCACTTACATGAA
AT TGGCCCTCAAATGIGICTGATACAACCCATGCTGCT TGTAACTATCGT TT TAAACTGCCAGGGTAA
ACTIGGACACACTTGAGCTAAGAAAAAGCT TT TAGATT TT TGCAAATTAATGTGAAAGATATGCTTTA
TGIGGATATAATATCTICTAAATTTCGGGGATGGTAGTCCTAGAAATGTAATCCTGCCCTAGCCGAGC
TTACCCTGCCAATAATTITTTACAGAATTGGTAAAACGGAGCACCTITTITTTGICCITGGCCACACT
GT TATCAACAGGGTGTAGAT TGACATCAATCTGTAGGIGTAAACCAGAAT TACTCT TTGTGACCACCA
GGAAATAGAGCAGTICAGTTCAGGGGITTCTTICTGTGAATTTAGCACTGTGACCTGCATACTACAAG
TCTACT =GT TT TCTATCCATTGT TTGTATCTGGGTAT TGCAAAAGGTAGGAAAAGGACCAACCAGAT
CAGCAGAGAAGAGT TGCCTIGGAGTT =CT TT TAGT TT TCTGCAGT TCAT TAGATAGTAACTAGGCCA
TGICATITTACTCCCTIGTAGTGAAGATATGTTGAAGTTGTACTGGTATACTCTICTACCITTCTGTA
AT TT TATATTGIGTAGACTTGATAAAAT TTATGIGICAATCACCACCATTAATATCAATATTGAGCCT
CAAT TCTTAT TT TICTGCCCAGIGGCTGCCAAAT TACTAACATT TACAATAATTCACTACTACTAAGA
TAATCTACTAGTTCGATCACATACTICAAATTGTTATGGAACTACTGICTICAGCATTGTGCTICTGA
TAACTGATAAGTATAATT TT TT TT TTGICCAGAGTGAACATGICTATTCT TCCACTGTACACACTAAT
AAAAGGAAAAATTGTAATATTGGGTAAATTCATGICCTTACACATGTAGTAGTTATGAGCCCATGICC
CTAGAATGAGTAATAATTTATCCCTCCCTIGGITGAATAGICAAGAATGCTGATTITAATTCTICTAA
CAGCTT TATCCCTCAGAAGGGAAGGCAAGCAAGT TATATATGTAGT TTAT TTGTAAGACTGATATGAA
AT TGGAAGATGAATCTACTATTAGCT TTAATTAT TT TTACAT TTAGGAATAT TGCATCAGTAACTCAT
AATITTGGITTICTGTTATCCTGAGTTAACACAAATTATCCAAGGAGATGGCGGATCATCTGCTITGA
GGIGTT TT TT TT TGAGAATT TTAATGTATCTGAATATAAAAGGTAAAAATATGCCAACTAGCAATTIC
TGCCCATTCCAGAAGT TTGGAAATAT TACTCATTACTAGGAATTAAATAAAATATGGT TTATCTATTG
TTATACCTCTTTTAATTCACATAGCTCATTTTTATCTTTTATTTTTGTTTGTTTTTTTTGAGATGGAG
TCTTGCTCTGICACCAGGCAGGAGTGCAGTGATGCAAATCTCGGCTCACTCTAGCCACCGACTCCCTG
GITCAAGCGATTCTCCTGCCTGAGCCTICTGAGTAGCTGGGATTACAGGCAGGCACCACCACGCCCAG
CTAATTITTGTAGAGACAGGATTICACCGTGTTGGCCAGGATGGICTCCATCTCCTGACCTCATGATC
TGCCTGCTICGGCCTCCCAAAGTGCTGGGATTACAGGIGGGAGCCACTACGCCIGGCCCACATAGCTC

AT TT TTAGACTCACTTCCAT TAAGTCTT GT TT GGACCCACGAACAT TGTCTT TT TT TT TT
TAAGATGG
AGTT TCACTT TT GT TGCCCAGACT GTAGTGCAAT GGTGCAATCTCAGCTCACTGCAATCTCT GCCTCC
TGGGTTCTAGCAATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGAATTACAGGCGCCCGCCACCACGCC
CAGCTAAT TT TT GT GT TT TTAGTAGAGACGGGGT TTCACCAT GT TGGGCAGGCCAGGGGT GATCCGCC
CACCTCAGCCTCCCAAAGTGCTGGGATTACAGGIGTGAGCCACCGCATCTGGCCAACATGICTITTIT
TT TT TT TICCIT TT TAACCACAAAGAGACT TAAGCAGTCCIT GTCACAGATGAT GAAT TGAT GT
TGCA
AGTATT GT CT TAGCTT GGAT TAAT TT TCTT GCTTACTGTAAT TT TAGATAATATAGCT TT
GTAATTAG
AGAT TT TATGTGTAAACCACAAAAAT GT TTACAT GAAGGCCATTAT TACAGATGTGACGT GCATAAT T
AT TAGTAATT TGTATGTT TACATGGGICAGICTGGCAAAAAATTAT GAAGTT TTAAAAAT TAAAAAAA
AT TATAAT GCCAGT TT TACT GGAAAGTAAAAT TATT TCAGTAATCGAT TATAGCAAAAGTAT TGATT T

TCATTCCAGACAAAAGTCAGAATGAAAGGTAATTTCTCAATACTCTTTCAGATTAATAAAAGTACCTG
TAGCGATT TT TATCAT TCACAAGTATATCACAAGTAAGTTAGAATT TGAGAACT GT GT TCTAGATCTC
TGAGGAGATGCAGICAGATTICTGAACTGICTCAGCAAATGGTAAGTAACTTAGAGCTAGTAATTAAT
AACCTGICCT TT GATT TCTGAT TCAGCCAAGAAT GGCCATAT TT GGGAAAGGCAGATCTGGAGAGTAA
CCACGTTTTCATTCATTTACCACTTCTAGGCCCCTCCAGAGCTCTCAGATATTTTGGGGTTGAGCCCT
TCCCCAAAGCCATACAGGACCT TT TT TT TGTGATCT GT TCTAGCCATT TT TATGTT GGGT GCTT
GTTA
TGGACTGAGCATTTATGICCTCCCACACCCCCCCCATACCITTITTGAAGTCCTAACCCCCAGTGTGA
TGGTAT TT GGAGACAGGGCCTT TGGAAGGTAATTACAGTTAGAAGAAGTCGGGAGGGT TGGGCCCAGG
TCTGAT TGGATTAGTGCCCT TATATGAAAAGACACCAGGACGGGCGCAGT GGCTCACACCTGTAATCC
CAGCACTITGGGAGGCCAAGGTGGGTGGATCACGAGGICAGGAGTTTGAGACCAGCCTGGCCAATGTA
GT GAAACACCATCTCTACTAAAAATACAAAAATTAGCT GGGT GT GGTAGCGGGCTCCT GTCATCCAAG
CTACTCGGGAGGGTGAGGCATGAGAATCACTTGAACCCGGGAGTTGGAGGITGCAGTGAGCCCAGATT
GIGCCACIGIACICCAGCCIGGGIGACAGAGIGAGACICIGICICAGAPPG
AGACACCAGAGAGCTT GT TAGAAGAGGTCATGTGAGCACACAGT TAGAAGACCT TCAAGCCAAAGAAG
AGGCCTGAGATTGAAACCTACCITGCAGGTACCITAATTITGGACTICCCAGCCTCCAAAACTGTGAG
AAATAAGTTICTGTTAAGTCACTCAGTCTGTGGTATTITGTTATGGCAGCCTGAGCAGGTAGTTGITC
TTTCAGAAGGTGTTGATAATAACCACATGCAACACCAAGTCACAAATAATAAAACAGATGTAACTTAT
AT TCATACAGAAAGTT GGGCACTGCCAT TGCCTT GT TGGT TTACACGGCT GT GCTAGT TCAGTAGCAG
AAAGGIGCTGGICTCCITTACTCAGTTTACAATCTAGGCAGTAGAATGTAATCACTGCTTTAAACTTG
ATACTGCTTAGGGAGAGAATCATTGGIGCTGGGTAACTITGGGITCTAGGITTACTITTTGIGTATAT
ATAACT GT TT TT GGTAAATCACAAGT =CT GGGCTT GTCGAATTAGAT TT TGTTACAGAT TATGAGCT
TTATTATGCTATACAGTTAGTTGTATGTATATATGCCTTTCCCACTAGATTTTAAGCTTTTTTTTTTT
TITTITTITTGTGACGGAGICTTGCTCTIGTCGCCCAGGCTGAAGTGGAGTGCAGTGGCACAATCTCG
GCTCACTGCAGCCTCCACCTCCTAGGTTCAAGCGATTCTCCTGCCTCGGCCTCCCAAGTAACTGGGAC
TACAGGCACGTGCCACCACACCCGGCTAATTTTTGTATTTTTTGTAGAGACAGGGTTTCGCCATGTTG
GCTAGGCTGGICTTGAACTICTGGCCTCAGGTGATCCACCCGCCTCAGCCTCCCAAAGTGCTGGGATT
TACAGGCATGAGCCACCACGCCCAGCTATAGCTCTITAAGGGITGTAAATTTATAATCATTCTITTAC
TCTCCTGCAAATTCTGTTGCACACTGCCITAATCAAGGTAGATGCTGAATGCATTITTGTATAATTGA
ATATGTTGCAATCCCCAACTCTCTCCAACTGITCCTGICAAAGCAGCCACTGGATTGTTAACTAATCC
ATAT TAGATGGGGT TAAT TAATATCAGATGGGACAAGTAAGGGCTAATAAGATTATAGGCCACCAAGT
AGATTTCTGTCTAGCTCTTATAGAGATTGAGTTTATTGGACCTGTTTGATAGGAAGTTTTGGTGTTTG
GGATGATTAAAACTGAAGTTCCTATTTATTGAATTATACCTATTTATATTATTICATATCAGTGGICC
ACATGCAAGTGAGGCTICTGAGACAGAGITTGAGTICTCTCTICAACTACCATAACACTTAACCIGTA

TCTT TT TT TT TT TT TT TT TT TT TAGACAGGAGTCTCGCTCTGTCACTCAGGCTGGAGT
GTAGTGGTAT
GATCTCGGCTCACTGTAACCTCTGCCTCCTGGATTCAAGCAGTTCTCCATGTCTCAGCCTCCCTAGTA
GCTGGGAT TACAGGCCTGTGCCACCATGCCTGGCTAAT TT TT TT TT TGTATT TT TAGTAGAGACGGGG
TT TTACCACGTT GGCCAGGCTGGTCTCGAACTCT TGACCTCGAGCGATCAACTT GCCT TGGCCTCCCA
AAGT GCTGGGAT TACAGGCATGAGCCACAGCGCCCAGCCGTCTT TT TT TT TAAATAGCAATT TAACAC
TGTTCACAGT TACTCATGTACATGTCAT GCCATCTATTACACTGTAAGTTCT GT GAGGGTAGCT GTAT
CAAATTTATCTAACTCTCTCTAGTATGCATGACATAGTAAGTATTCAATAAATATTTGCATATTAGTG
ATAAGGATACAGGTTCTGAATAGTGGGTCCTTACCATTTAAGAATTAGTATTTGATGGCCGGGCGGGG
TGGCTCACGCCT GTAATCCCAGCACT TT GGGAGGCT GAGGCGGGCGGATCAT GAGATCAGGAGATCGA
GACCATCCTGGCTAACATGGTGAAATCCCGTCTTTAC
TACAAAAGAATTAACCAAGTGTG
GTGGTGGGTGCCTGTAGTCCCAGCTACTGCTTTGTGAGGCTGAGGCAGGCAGATCACCTGAGGTGGGA
AATTCAAGACCAGCCTGACCAACATGGAGAAACCCCATCTCTACTAAAAATACAAAATTAGCCGGGCG
TGGTGGCGCATGTCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTGAACCCGGGAG
GCGGAGCTTGCAGTGAGCCAGGATCGCGCCACTGCACTCCAGCCTGGGCGACAGAGCGAGACTCCGTC
TC
TTAGTATT TGATAT TT GATCAT TAAATATGAATTAAGAGGACT T
AGACTT TT TGTTAAAT GTCAAGCT GGGAAAAGTT GTCATT TAAATGAATT GCCTCT TATT TAAT TTCG

TCTGATGATACATTTTGTTTTTATTTTGTAAAAAATTATTTTTTTTCTTTTTGGAGACAGGGTCTTGC
TCTGTTGCCCAGGCTGGTCACAAACTCCTGACCTCAAGCAATCCTCCTGCCTTAGCCTCCCAAAATGC
TGGGAT TACAGGCGTGACGACCTCGCCCGGCCTT GTAT TATGATACAT TT TGAACAACTACAAGTAGA
CTTGGTATAATGAACCTGCACGTACCCATTGCCAAGTTCTGACAACTGTCTGTCTATAGCCAATTATG
CATTTCTTAAATTAGAACCCCCCCAATATACCCAAATATATATATATGTGTGCATATATATAGTAAGT
TGTAACAAAGTT GT GAAT TCATACCT GAAGTATCTCAAGT GATGCAAGTT TTAT GAAT TT TT GT
TTAT
GCCTTTTGGGAAGAGTTGTATTGACAAATTTTTTATGCTTAAAGTAAACCATAAATCAAAAAAATAAA
ATCTAGGATGCAATAAAACAAAACAACTTCTTGACATAAGTATGGTATGTAAATCTGTTTTGATTGGA
AATCAATTTGTTATATTGCCAGAATTCCTGTTTTAGAATACATCTCTGCTGATCTGTCTGTATTCTTA
GACT GCATATCT GGGATGAACTCT GGGCAGAATTCACATGGGCT TCCT TT GAAATAAACAAGACTTT T
CAAATTCT TAGTCGATCT GCAGAACCTGTAGCCAGGCACT GAACCATT TT GATAGATGCAGTAATCGT
TGCAAGTGTATATTTCAAGGGAGTTCTGGCTGGGTCCTAGTTTATGCTTGTGGCAGAAGCAGTGAGTA
ACTGGGAGGAAGTTGGTGAGTAAGCTTCAAGGAAGAAGTCATTTTTAGTACTCTGGATCTTCCTGATT
TTAAAGCACTACAAAATGGT GCAT TT TCAT TCTT GTCAAGTGATAACAGATATATTCT GATGAGCCT G
AAAT GAATATATAT TGTATCAT TT TTATAATATCTAGCAAGGTT TGTATT TTCCTAGAACTT GAACTA
AATT TCAGTTCATAAAAT TTATAAAATACT TAGT TGTT GTAAAATATT TT TGGAAT GT TCACATAGGT
GACACACAAATGTCCCAT TT TCAT TCTT TCTATAGTAAATAT GT TCTGATAT GT GAAGGT TTAGCAGA
TGCATCAGCATT TAATCCTAGAGGATCT GGCATAATCT TT TCCCCCAAGAATAGAAAT TT TT TCTGCT
TATGAAAGTAGTACAT GT TTCT TTAAAAACAAATCAATAT TGACTTCT GCCT GCTGTATAGCACTAT G
CCTCCACCTGGCCATGACCAGGGGCATGTCCTGGTCCACCTACCTGAAAATGTTTGCAACCAGCCTCC
TGGCCATGTGCACAGGGGCTGAAGTTGTCCCACAGGTATTACGGGCCAACCTGACAATACATGAAGTT
CCACCAAAGTCTGAGAACTCAGAACTGAGCTTTGGGGACTGAAAGACAGCACAAACCTCAAATTTCTC
AGCACTGGAAACCTCAAAATATAACTGAATTCCATAAATAAGATTTTAAGTCTTAAATATGTATTTTT
AAATGTATTAAAAGTCAAGCTGCTTGTATTTAAGCACCTAATACAATGCTTAGGTTGTAAAAGGAGAT
GCTCAATAGGTACTAACT GATATATT GAGATT TAAT TATGGT TT GACCAATATT TATT GGAAACCGCC
AAAGCT TAAATCATCAGCTTCT TGAATGTGAT TT GAAAGGTAAT TTAGTATT GAATAGCATGTGAGCT
AGAGTATTTCATTCTTTCTGGTTTATTTCTTCAAATAGACTTTGAATATAATGGTGAATGGGTATTAT

AAAT TAACTAATAAAAAT GACATT GAAAAT GAAAAAAT AT AT AT AT TAAAGT GT AGAAAGT GAC
CAGG
CGTGGIGGCTCACACCIGTAATCCAAGCACCTIGGGAGGCTGAGGCAGGAGGATCTCTTGATCCCAGG
AGTTCAAGACCAGCCTGGGCAACATAGCGAGACTTCGTCTCT
GAGAGAGAAAAAAA
TT TT TT TTAT TTAAAAAAAGIGTAGAAAGTGICAAGACCCCACT TCTTACCATTAT TIGGTATATTIC
TCTATACCCACCCACCCTTCCTCCTTACTCCCTCCCTCCCTTCCCAATCTTTTTATCTTTTTGTATTC
TGAT TT TT TGIT TGTATATT TIGCTT TART TTAATGTATCCT TTAAAAAT TTCCCATACATT TTATAT

GTATATATAAAAACGCATGCTGCCAAAGATAATTTATAAGAAAGACCATTGAATTTTTTTAAAAGTGA
TATATATTCATTGAAAAAAATT TAGAATATATAGCAAAGCAATAAAGAACTAAATAAAAT TGCTGTAA
CTCCTCTT TCAAAGATAAGTGCTT TTATGATT =GT TGTATT TT TT TCTGTATATAGGTACATATATA
GTATTTATAAAGCTGTACTCATAGTACATTITCACATCACAGGTACCATATCAGTGTTATTAAATATT
TIGTATGCCAGGGGCTAGACATACCAAGACAACCAATATGIGGITCTACTTAAATAATATTAGAGTAT
CT TT TATGATGACACT TCATGAGT TGACTATAATAATCTTAGACTICTAAGAGT TIGGGT TT TCAAAA
GATCACTTAGCT IT IT TGGGTGAT IT TTCCCCCT TACTGTGAGATGAGAGAGGCTGTT TGGATT TGGG
AT TGGGGTAGCGGGGACAGCAACT TT TCTT TT CT TT TT CT TT TT TATT TTGAGGTAGGGTAT
TGCTGT
GTCACCCAGGCTGGAGTGCAGTGGTGTGATCTCGGCTCACTGCAACCTCCACCTCCCGGGCTCAGGTG
ATCCTCCTGCTICAGCCTCCCAGTAACTGGGACTACAGGCGCGTGCCACATGCCIGGCTAATTITGTA
TT TT TAGTAGAGATGGGGTT TCACCATGTIGGCCAGGCTGGICTCTAACTCCTGACCTCAGGTGATAC
GCCCACCIGGGCCTCCCAAAATACTGGGATTACAGGCATGAGCCGCTGCATCAGCCAGCAGTITTICT
TGTGGTTTTTTTTGTTTGTTTTGTTTTGTTTTGTTTTTGAGATAGGGTCTTACTCTGTTGTCCACGCT
GGAGTGCTGIGGTATGATCGTAGCTCACTGCAGCCTCAAACTCCIGGGCTCAAGTGATTCCTICTGCC
TCCGCCTCCCGAGTAGCTGGGACTACAGGTATGCACCACCATACCIGGCAAATT TT TACAAAGTITT T
TGTAGGGACGGGGICTTGCTACATTCCCCATGICGGICTTGAACTCCTGGCCTCAAGCAACTCTCCTG
TCTCAGCCTCCCAAAGCACTGGGATTACAAGIGTGAGCCACCACACCATGCCAGTT TT TCCTGT TCAG
TGTGATATITTATCTIGTTAGACTACAGTGIGTTAAAACTIGTITTACTAAATTITCAAACATACTCA
AAAGTGGAGAGAATAGTATAAT GAAT ACCC GT AT GT T CAT CACCCAT GT T TAGAAT AT TAT T
AAATAT
AAAGATITTGCTGCGITTGICTTAGCTCTITAAAATTITTCTITTICTCTITGTGACCTAAAGGAAAT
TCCATATCTTATCACTITACTICTACATTCTTGACTAAGATGACTAAGACATATAGTTACATGGITTT
TTGITTIGTTITTGITTITTAAAGACGAAATCTCGCTCTIGTCCCCCAGGCTGGAGTGCAATGGIGCC
ATCTCAGCTCAGTGCAACCTCTGCCTTCTGGGTACAAGCGATTCTCCTGCCTCAGCCTCCCAAGTAGC
TGGGATTACAGGCTCCTGCCACCACGCCTGGCTAATTTTTGTATTTTTAGTAGAGACGGCGGGGGGAG
GITTCACCATGTTGACAAGGCTGGICTGGAACTCCTGACCTCAGGTGATCCACCCGCCTCGGCCTCCC
AAAGTGCTGGGATTACAGGCGTGAGCCACCGCGCCCAGCCIGTITTITTGITTGIGTGITTIGTTITT
TTTGAGACAGAGTCTTGCTCTGTTTCCCAGGCTGGAGTGAAGTGGTGCCATCTCAGCTCAGAGACAGA
GICTTGCTCTGITTCCCAGGCTGGAGTGAAGIGGIGCCATCTIGGCTCACTGCAACCTICACCTCCCA
GGTTCAAGTGATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGACTACAGGCATGTGTCACCACACCCG
GCTAATTTTTTTGTATTTTTAGTAGAGACGGGATTTCACCGTGTTGCCCAGGCTGGTCTCGAACTCCT
GAGCTCAGGCAGICTGCCTGCCTCAGCCTCCCAAAGTGCTGGGATTACACGTGTGAACCAACCCGCCC
GGCCTGTTGT TT TCTTACATAATTCATTATCATACCTACAAAGT TAACAGTTACTAATATCATCTTAC
ACCTAAATTICTCTGATAGACTAAGGITATTITTTAACATCTTAATCCAATCAAATGITTGTATCCTG
TAATGCTCTCAT TGAAACAGCTATAT =CT TT TTCAGATTAGTGATGATGAACCAGGT TATGACCTIG
ATTTATITTGCATACCTAATCATTATGCTGAGGATTIGGAAAGGGIGITTATTCCTCATGGACTAATT
ATGGACAGGTAAGTAAGATCTTAAAATGAGGT TT TT TACT TT =CT TGIGTTAATT TCAAACATCAGC
AGCTGTICTGAGTACTTGCTATTTGAACATAAACTAGGCCAACTTATTAAATAACTGATGCTITCTAA

AATCTTCT TTAT TAAAAATAAAAGAGGAGGGCCT TACTAATTACTTAGTATCAGTT GT GGTATAGTGG
GACT CT GTAGGGACCAGAACAAAGTAAACATT GAAGGGAGAT GGAAGAAGGAACTCTAGCCAGAGTCT
TGCATTTCTCAGTCCTAAACAGGGTAATGGACTGGGGCTGAATCACATGAAGGCAAGGTCAGATTTTT
AT TATTAT GCACATCTAGCT TGAAAATITICT GT TAAGTCAATTACAGTGAAAAACCT TACCTGGTAT
TGAATGCTTGCATTGTATGTCTGGCTATTCTGTGTTTTTATTTTAAAATTATAATATCAAAATATTTG
TGTTATAAAATATTCTAACTATGGAGGCCATAAACAAGAAGACTAAAGTTCTCTCCTTTCAGCCTTCT
GTACACATTTCTTCTCAAGCACTGGCCTATGCATGTATACTATATGCAAAAGTACATATATACATTTA
TATT TTAACGTATGAGTATAGT TT TAAATGTTAT TGGACACT TT TAATAT TAGT GT GTCTAGAGCTAT
CTAATATATTTTAAAGGTTGCATAGCATTCTGTCTTATGGAGATACCATAACTGATTTAACCAGTCCA
CTATTGATAGACACTATTTTGTTCTTACCGACTGTACTAGAAGAAACATTCTTTTACATGTTTGGTAC
TT GT TCAGCT TTAT TCAAGT GGAATT TCTGGGTCAAGGGGAAAGAGTT TATT GAATAT TT TGGTATT
G
CCAAAT TT TCCTCTAAGAAGTT GAATCATT TTATACTCCT GATGTTATAT GAGAGTACCT TTCTCTTC
ACAATT TGTCTCTT TT TT TT TT TT TT TT GAGACAAGGTCTCT GT TGCCCAGGCT GGGGTGCAGT
GCAG
CAGAATGATCACAGTTCACTGCAGTCTCAACCTCCTGGGTTCAAGCGATCCTTCCACCTCAGCCTCCT
GAGTAGCT GGGACTATAGGT GT GCGCCACCACTCCCAGCTAATATT TT TATT TT GTAGAAACAGGGT T
CGCCAT GT TACCCAGCCTCCCAAAGT GCTGGGAT TACAGGCATGAGCCACTGGCCCAGTT TCTACAGT
CTCTCT TAATAT TGTATATTATCCAGAAAATT TCAT TTAATCAGAACCTGCCAGTCTGATAGGT GAAA
AT GGTATCTT GT TT TTAT TT GCAT hA
TTAT GATAGT GGTATGCT TGGT TT TT TT GAAGGT
ATCAAATTTTTTACCTTATGAAACATGAGGGCAAAGGATGTGATACGTGGAAGATTT
TT T
TTAATGCATT TT TT TGAGACAAGGTCTT GCTCTATT GTCCAGGCTGGAGT GCAGTGGCACAATCACAG
TTCACTCCAGCCTCAACATCCT GCACTAAAGT GATT TTCCCACCTCACCTCTCAAGTAGCTGGGACTA
CAGGTACATGCTACCATGCCTGGCTAAT TT TT TT TT TT TT GCAGGCAT GGGGTCTCACTATATT GCCC
AGGT TGGT GT GGAAGT TTAATGACTAAGAGGT GT TT GT TATAAAGT TTAATGTATGAAACTT TCTAT
T
APT TCCT GATT TTAT TI CT GTAGGACT GAACGTCT TGCTCGAGAT GT GATGAAGGAGAT GGGAGGCC
ATCACATTGTAGCCCTCTGTGTGCTCAAGGGGGGCTATAAATTCTTTGCTGACCTGCTGGATTACATC
AAAGCACT GAATAGAAATAGTGATAGATCCAT TCCTAT GACT GTAGAT TT TATCAGACTGAAGAGCTA
TT GT GT GAGTATAT TTAATATATGAT TCTT TT TAGT GGCAACAGTAGGTT TI CT TATATT TI CT
TTGA
ATCTCTGCAAACCATACTTGCTTTCATTTCACTTGGTTACAGTGAGATTTTTCTAACATATTCACTAG
TACT TTACATCAAAGCCAATACTGTT TT TT TAAAACTAGTCACCTT GGAGGATATATACT TATT TTAC
AGGTGTGTGTGGTTTTTTAAATAAACTCCTTTTAGGAATTGCTGTTGGGACTTGGGATACTTTTTTCA
CTATACATACTGGTGACAGATACCCTCTCTTGAGCTACATCGGTTTGTGGGGAGTCAAAAGTCCTTTG
GAGCTAGGTTTGACAAATAAGGTGGGTTAACACTTGTTTCCTAGAAAGCACATGGAGAGCTAGAGTAT
TGGCGAAT TGAAGAAATCCCCCTT TT TT TT TAACACACTTAAGAAAGGGGACTGCAGGTATACTCAAG
AGAGTAAGTCGCACCAGAAACCACTT TT GATCCACAGTCT GCCT GT GTCACACAAT TGAAAT GCATCA
CAACATTGACACTGTGGATGAAACAAAATCAGTGTGAATTTTAGTAGTGAATTTCATTCATAATTTGA
TCGTGCAAACGTTTGATTTTTATTACTTTAGACTATTGTTTCTGATTTTATGTTGGGTTGGTATTTCC
TGTGAGTTACTGTT TTACCT TTAAAATAGGAATT TT TCATACTCTTCAAAGATTAGAACAAATGTCCA
GTTTTTGCTGTTTCATGAATGAGTCCTGTCCATCTTTGTAGAAACTCGCCTTATGTTCACATTTTTAT
TGAGAATAAGACCACT TATCTACATT TAACTATCAACCTCATCCTCTCCATTAATCATCTAT TT TAGT
GACCCAAGTTTTTGACCTTTTCCATGTTTACATCAATCCTGTAGGTGATTGGGCAGCCATTTAAGTAT
TATTATAGACAT TT TCACTATCCCAT TAAAACCCTT TATGCCCATACATCATAACACTACTTCCTACC
CATAAGCTCCTTTTAACTTGTTAAAGTCTTGCTTGAATTAAAGACTTGTTTACGGTATCGATAAGCTT
GATATCAAAACGCCAACT TT GACCCGGAACGCGGAAAACACCTGAGAAAAACACCT GGGCGAGTCTCC

ACGTAAACGGTCAAAGTCCCCGCGGCCCTAGACAAATATTACGCGCTATGAGTAACACAAAATTATTC
AGATTTCACTTCCTCTTATTCAGTTTTCCCGCGAAAATGGCCAAATCTTACTCGGTTACGCCCAAATT
TACTACAACATCCGCCTAAAACCGCGCGAAAATTGTCACTTCCTGTGTACACCGGCGCACACCAAAAA
CGICACTITTGCCACATCCGICGCTTACATGIGTICCGCCACACTIGCAACATCACACTICCGCCACA
CTACTACGTCACCCGCCCCGTTCCCACGCCCCGCGCCACGTCACAAACTCCACCCCCTCATTATCATA
TTGGCTTCAATCCAAAATAAGGTATATTATTGATGATGTTTAAACATTAAGAATTAATTCGATCCTGA
ATGGCGAATGGACGCGCCCIGTAGCGGCGCATTAAGCGCGCGGGIGIGGIGGITACGCGCAGCGTGAC
CGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCG
CCGGCTITCCCCGICAAGCTCTAAATCGGGGGCTCCCITTAGGGITCCGATTTAGAGCTITACGGCAC
CICGACCGCAAAAAACTIGATTIGGGIGATGGITCACGTAGIGGGCCATCGCCCTGATAGACGGITTT
TCGCCCITTGACGTIGGAGICCACGTICITTAATAGIGGACTCTIGTICCAAACTGGAACAACACTCA
ACCCIATCGCGGICTATICTTITGATTTATAAGGGATGITGCCGATTICGGCCIATTGGITAAAAAAT
GAGCTGATTTAACAAAAATITTAACAAAATICAGAAGAACTCGICAAGAAGGCGATAGAAGGCGATGC
GCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGICAGCCCATTCGCCGCCAAGCTCT
TCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTC
GATGAATCCAGAAAAGCGGCCATTITCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGICACGA
CGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGA
TGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCG
ATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAG
CCATGAIGGATACTITCTCGGCAGGAGCAAGGIGAGATGACAGGAGATCCIGCCCCGGCACTICGCCC
AATAGCAGCCAGICCCTICCCGCTICAGTGACAACGICGAGCACAGCTGCGCAAGGAACGCCCGICGT
GGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGA
CAAAAAGAACCGGGCGCCCCIGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGICTGT
TGIGCCCAGICATAGCCGAATAGCCICTCCACCCAAGCGGCCGGAGAACCIGCGTGCAATCCATCTIG
TICAATCATGCGAAACGATCCICATCCIGICICTIGATCAGAGCTIGATCCCCIGCGCCATCAGATCC
TIGGCGGCAAGAAAGCCATCCAGITTACTITGCAGGGCTICCCAACCITACCAGAGGGCGCCCCAGCT
GGCAATTCCGGITCGCTIGCTGICCATAAAACCGCCCAGICTAGCTATCGCCATGTAAGCCCACTGCA
AGCTACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCC
GGGGICAGCACCGTTICTGCGGACTGGCTITCTACGTGAAAAGGATCTAGGIGAAGATCCITTITGAT
AATCTCATGGCTGCAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGC
TICCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGITGCAGGACCACTICTGCGCTCGGCCC
TICCGGCTGGCTGGITTATTGCTGATAAATCTGGAGCCGGIGAGCGIGGGICTCGCGGIATCATTGCA
GCACTGGGGCCAGAIGGTAAGCCCICCCGTATCGTAGITATCTACACGACGGGGAGICAGGCAACTAT
GGATGAACGAAATAGACAGATCGCTGAGATAGGIGCCICACTGATTAAGCATIGGTAACTGICAGACC
AAGITTACTCATATATACTITAGATTGATTTAAAACTICATTITTAATTTAAAAGGATCTAGGIGAAG
ATCCITTITGATAATCTCATGACCAAAATCCCITAACGTGAGITTICGTICCACTGAGCGICAGAC
RightITR = first underlined and bold sequence U6 = first underlined sequence CMV = first bold sequence dCas9VP64 = second underlined sequence HGHpA = second bold sequence EFla = third underlined sequence MPH = third bold sequence HGHpA = fourth underlined sequence Packaging Signal = fourth bold sequence LeftITR = second underlined and bold sequence SEQ ID NO:9 LV- SAM
L e ftL TR-P ack agi ng S i gnal -RRE-U6jaMIN-CMV-dC a s 9 VP 64 -HGHp A-EF 1 a-MPH-HGHpA-Ri ghtLTR
AGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGAC
GTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAA
ATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATG
AGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCA
CCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAAC
TGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACT
TTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGTCGCCG
CATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCA
TGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTG
ACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCT
TGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAG
CAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTA
ATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTT
TATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATG
GTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGA
CAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATAT
ACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATC
TCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAA
GGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACC
AGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAG
CGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCA
CCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCT
TACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGT
GCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAA
AGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGA
GCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCT
GACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCG
GCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGA
TTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTCAGATGGTCCCCAGATATGGCCCAACCCTCA
GCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCTCCCCCAAGGACCTGAAATGACCCTGCGCCTTAT
TTGAATTAACCAATCAGCCTGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAG

AGCTCACAACCCCTCACTCGGCGCGCCAGTCCTCCGACAGACTGAGTCGCCCGGGGGGGATCACCAGA
TACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAA
TACGCAAACCGCCT CT CCCCGCGCGT TGGCCGAT TCAT TAAT GCAGCT GT GGAATGTGTGTCAGT TAG
GGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCA
ACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTC
AGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCAT TCTC
CGCCCCAT GGCT GACTAAT T TI TI T TAT T TAT GCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTAT T
C
CAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTT TTGCAAAAAGCT TGGACACAAGACAGGCT T
GCGAGATATGTT TGAGAATACCACTT TATCCCGCGTCAGGGAGAGGCAGTGCGTAAAAAGACGCGGAC
TCAT GT GAAATACT GGT T TT TAGT GCGCCAGATCTCTATAAT CT CGCGCAACCTAT TT
TCCCCTCGAA
CACT TT TTAAGCCGTAGATAAACAGGCTGGGACACT TCACATGAGCGAAAAATACATCGTCACCTGGG
ACAT GT TGCAGATCCATGCACGTAAACT CGCAAGCCGACT GATGCCT T CT GAACAATGGAAAGGCAT T
AT TGCCGTAAGCCGTGGCGGTCTGTACCGGGTGCGT TACT GGCGCGTGAACT GGGTAT TCGTCATGTC
GATACCGT TTGTAT TTCCAGCTACGATCACGACAACCAGCGCGAGCTTAAAGTGCTGAAACGCGCAGA
AGGCGATGGCGAAGGCTTCATCGT TAT T GATGACCT GGTGGATACCGGTGGTACTGCGGT TGCGATTC
GT GAAATGTATCCAAAAGCGCACT T T GT CACCAT CT TCGCAAAACCGGCTGGTCGTCCGCTGGT TGAT
GACTAT GT TGTTGATATCCCGCAAGATACCTGGATTGAACAGCCGTGGGATATGGGCGTCGTAT TCGT
CCCGCCAATCTCCGGTCGCTAATCTT T T CAACGCCT GGCACT GCCGGGCGT T GT TCTT TT TAACTTCA

GGCGGGTTACAATAGT T T CCAGTAAGTAT T CT GGAGGCTGCATCCATGACACAGGCAAACCT GAGCGA
AACCCT GT TCAAACCCCGCT TTAAACATCCTGAAACCTCGACGCTAGTCCGCCGCT TTAATCACGGCG
CACAACCGCCTGTGCAGT CGGCCCT T GATGGTAAAACCAT CCCT CACT GGTATCGCAT GAT TAACCGT
CT GATGTGGATCTGGCGCGGCAT T GACCCACGCGAAAT CCTCGACGTCCAGGCACGTAT T GT GATGAG
CGAT GCCGAACGTACCGACGAT GAT T TATACGATACGGTGAT TGGCTACCGTGGCGGCAACTGGATT T
AT GAGT GGGCCCCGGATCT T TGTGAAGGAACCT TACT T CT GT GGTGTGACATAAT T
GGACAAACTACC
TACAGAGATT TAAAGCTCTAAGGTAAATATAAAATT TT TAAGTGTATAAT GT GT TAAACTACTGATTC
TAAT TGTT TGTGTATT TTAGAT TCCAACCTATGGAACTGATGAATGGGAGCAGTGGTGGAATGCCTT T
AATGAGGAAAACCT GT TT TGCTCAGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACTCTCA
ACAT TCTACTCCTCCAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACT TTCCTTCAGAAT TGCTAA
GT TTTT TGAGTCATGCTGTGTT TAGTAATAGAACTCTTGCTTGCTT TGCTAT TTACACCACAAAGGAA
AAAGCTGCACTGCTATACAAGAAAAT TATGGAAAAATAT T CT GT AACCT T TATAAGTAGGCATAACAG
T TATAATCATAACATACT GT TTTT TCT TACTCCACACAGGCATAGAGT GT CT GCTAT TAATAACTAT G

CT CAAAAAT T GI GTACCT TTAGCT TT TTAATT TGTAAAGGGGTTAATAAGGAATAT TTGATGTATAGT
GCCT TGACTAGAGATCATAATCAGCCATACCACATT TGTAGAGGTT T TACT T GCT T TAAAAAACCTCC
CACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAAT TGT T GT TGTTAACT TGTT TAT T GCAGCT
TATAATGGTTACAAATAAAGCAATAGCATCACAAAT TTCACAAATAAAGCAT TTTTTTCACTGCATTC
TAGT TGTGGT TI GI CCAAACTCAT CAAT GTAT CT TATCAT GI CT GGAT
CAACTGGATAACTCAAGCTA
ACCAAAATCATCCCAAACTTCCCACCCCATACCCTATTACCACTGCCAAT TACCTGTGGT T T CAT T TA
CT CTAAACCT GI GAIT CCTCTGAAT TAT TT TCAT TT TAAAGAAATTGTAT TI GI
TAAATATGTACTAC
AAACTTAGTAGT TGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCT TGATCTGTGGATCTAC
CACACACAAGGC TACT TC CC TGAT TAGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCAC TGAC
CT TTGGATGGTGCTACAAGC TAGTACCAGT TGAGCCAGATAAGGTAGAAGAGGCCAATAAAGGAGAGA
ACACCAGC TTGT TACACC C TGTGAGC C TGCATGGGATGGATGAC CC GGAGAGAGAAGTGT TAGAGTGG

AGGT TTGACAGCCGCC TAGCAT TTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACT TCAAGAACTG

CTGATATCGAGC TTGC TACAAGGGAC TT TC CGC TGGGGAC TT TC CAGGGAGGCG TGGC C TGGGC
GGGA
C TGGGGAG TGGC GAGC CC TCAGATCC TGCATATAAGCAGC TGC T TT TTGC C TGTAC TGGGTC TC
TCTG
GT TAGACCAGATC TGAGC C TGGGAGC TC TC TGGC TAAC TAGGGAACCCAC TGCTTAAGCC
TCAATAAA
GC TTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTC TGTTGTGTGACTCTGGTAAC TAGAGATC CC TC
AGAC CC TT TTAG TCAG TG TGGAAAATC TC TAGCAGT GGCGCCCGAACAGGGACT TGAAAGCGAAAGGG
AAACCAGAGGAGCT CT CT CGACGCAGGACT CGGCTT GC T GAAGC GC GCAC GGCAAGAGGC GAGGGGC
G
GC GACT GGTGAGTACGCCAAAAAT TT TGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGCGAGAGCG
T CAG TAT T AAGC GG GGAGAAT T AGAT C G C GAT GGGAAAAAAT
TCGGTTAAGGCCAGGGGGAAAGAAAA
AATATAAATTAAAACATATAGTAT GGGCAAGCAGGGAGCTAGAACGAT T C GCAGT TAAT C CT GGCCT G
TTAGAAACAT CAGAAG GC T GTAGACAAATACT GG GACAGC TACAAC CAT C CC T T CAGACAGGAT
CAGA
AGAACT TAGAT CAT TATATAAT ACAG TAGCAACC CT CTAT T GT GT GCAT CAAAG
GATAGAGATAAAAG
ACACCAAGGAAGCT TTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCG
GC CGGC CGCT GAT C T T CAGACCTGGAGGAGGAGATATGAGGGACAATT GGAGAAGT GAAT TATATAAA

TATAAAGT AG TAAAAAT T GAAC CAT T AG GAGT AG CAC C CAC CAAGG CAAAGAGAAGAG T G
GT GCAGAG
AGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTTGGGAGCAGCAGGAAGCACTATGG
GC GCAGCG TCAATGAC GC TGACGGTACAGGCCAGACAATTATTGTC TGGTATAGTGCAGCAGCAGAAC
AATTTGCTGAGGGC TATTGAGGCGCAACAGCATC TGTTGCAACTCACAGTCTGGGGCATCAAGCAGC T
CCAGGCAAGAATCC TGGC TGTGGAAAGATACC TAAAGGATCAACAGCTCC TGGG GAT T T G GG GT T
GC T
CT GGAAAACT CAT T TGCACCACTGCT GT GC CT TGGAAT GC TAGT
TGGAGTAATAAATCTCTGGAACAG
AT TT GGAAT CACAC GAC C T G GAT G GAGT GGGACAGAGAAATTAACAAT TACACAAGCT
TAATACACT C
CT TAAT TGAAGAAT C G CAAAAC CAGCAAGAAAAGAAT GAACAAGAAT T AT TGGAAT
TAGATAAATGGG
CAAGTT T GT GGAAT TGGT TTAACATAACAAAT T GGC T GT GGTATATAAAAT TAT
TCATAATGATAGTA
GGAGGCTT GGTAGGTT TAAGAATAGT TTTT GC T GTACT TT CTATAGTGAATAGAGT TAGGCAGGGATA
TT CACCAT TAT C GT TT CAGACC CACC T C CCAACC CC GAGGGGAC CC GACAGGCC
CGAAGGAATAGAAG
AAGAAG GT GGAGAGAGAGACAGAGACAGAT CCAT T C GAT TAGT GAACGGAT C T C GACGGT AT
CGCCAA
AT GGCAGTAT T CAT CCACAATT TTAAAAGAAAAGGGGGGATT GGGGGGTACAGT GCAGGGGAAAGAAT
AG TAGACATAAT AG CAACAGACAT ACAAAC TAAAGAAT TACAAAAACAAATTACAAAAAT TCAAAAT T
TT CGGGTT TAT TACAGGGACAGCAGAGAT C CAGT TT GGAT CGATAAGCTT GATATCGAAT TCGTAGGG

ATAACAGGGTAAT GAGGGCC TAT T TCCCAT GATT CC T T CATATT TGCATATACGATACAAGGCT GT
TA
GAGAGATAAT TGGAAT TAAT TT GACT GT AAACACAAAGAT AT TAGT ACAAAATAC G T GAC GT
AGAAAG
TAATAAT T TCTT GGGTAGTT TGCAGT TT TAAAAT TAT= TTAAAATGGACTAT CATATGCT TACCGT
AACT TGAAAGTATT T C GAT T TCTT GGCT TTATATAT CT TGIGGAAAGGACGAAACACCGNOMONN
iiitiggi4NMANGTT TTAGAGCTAGGCCAACAT GAGGAT CACC CAT GTC T GCAGGGC CTAGCAAGT
TAA
AATAAGGCTAGT CC GT TAT CAACT TGGCCAACAT GAGGAT CACC CAT GTC T GCAGGGC
CAAGTGGCAC
CGAGTCGGTGCT TTTTTT GGAT CC T GT T GACAAT TAAT CAT C GGCATAGTATAT
CGGCATAGTATAAT
AC GACAAGGT GAGGAACTAAAC CAT GGC CAAGT T GACCAGT GCC GT TCCGGT GC T CAC
CGCGCGCGAC
GT CGCCGGAGCGGT CGAGTT CT GGACCGACCGGCTCGGGT TC TC CC GGGACT T C GT
GGAGGACGACT T
CGCC GGT GT GGT CC GGGACGAC GT GACC CT GT T CAT CAGC GC GGT C CAGGAC CAGGT GGT
GC CGGACA
ACAC CC T GGC CT GGGT GT GGGT GC GC GGCC T GGACGAGCT GTAC GC CGAGT GGT CGGAGGT
C GT GT C C
AC GAAC T T CC GGGACGCC T C CGGGCC GGCCAT GACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGT
T
CGCC CT GC GC GACC CGGC CGGCAACT GC GT GCACTT CGTGGCCGAGGAGCAGGACT
GATAGGGATAAC
AG GG TAAT GC TAGCATAG TAATCAAT TACGGGGTCATTAG TTCATAGC CCATATATGGAG TTCC GC
GT
TACATAAC TTAC GG TAAATGGC CC GC C TGGC TGACC GC CCAACGAC CC CC GC CCAT TGAC
GTCAATAA

TGACGTATGTTCCCATAGTAACGTCAATAGGGAC TT TC CATTGACG TCAATGGG TGGAGTAT TTACGG
TAAACTGCCCAC TTGGCAGTACATCAAG TG TATCATATGC CAAG TACGCC CC C TAT TGAC GTCAATGA

CGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACA
TC TACG TATTAG TCATCGC TAT TACCATGG TGATGC GG TT TTGGCAGTACATCAATGGGC GTGGATAG
CGGTTTGACTCACGGGGATTTCCAAGTC TCCACCCCAT TGACGTCAATGGGAGT TTGT TT TGCACCAA
AATCAACGGGAC TT TC CAAAATGTCG TAACAAC TCC GC CC CATTGACGCAAATGGGCGGTAGGC GTG T

AC GG TGGGAGGTC TATATAAGCAGAGC TCG TT TAGTGAAC CG TCAGATCGCC TGGAGACGCCATCCAC
GC TG TT TTGACC TCCATAGAAGACACCGGGACCGATCCAGCC TC CGCGGATTCGAATC CC GGCC GGGA
AC GG TGCATTGGAACGCGGATTCC CC GTGC CAAGAG TGAC GTAAGTAC CGCC TATAGAGTCTATAGGC
CCACAAAAAATGCTTTCTTC TT TTAATATACT TT TT TGTT TATC TTATTTCTAATACTTTCCCTAATC
TC TT TC TT TCAGGGCAATAATGATACAATG TATCATGC C TC T TTGCAC CATTC
TAAAGAATAACAGTG
ATAATTTCTGGGTTAAGGCAATAGCAATATTTCTGCATATAAATATTTCTGCATATAAATTGTAACTG
ATGTAAGAGG TT TCATAT TGC TAATAGCAGC TACAATC CAGC TACCATTC TGC T TT TATT
TTATGGT T
GGGATAAGGC TGGATTATTC TGAG TC CAAGC TAGGC CC TT TTGC TAATCATGTTCATACC TC TTATC
T
TCCTCCCACAGC TCCTGGGCAACGTGCTGGTC TGTGTGCTGGCCCATCAC TT TGGCAAAGAATTGGGA
TCGT AC GGCCAC CAT GAAAAGGCC GGCGGC CACGAAAAAGGC CGGC CAGGCAAAAAAGAAAAAGGACA
AGAAGTACAGCATCGGCCTGGCCATCGGCACCAACT CT GT GGGCTGGGCCGT GATCACCGACGAGTAC
AAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGAT
CGGAGCCCTGCT GT TCGACAGCGGCGAAACAGCCGAGGCCACCCGGCT GAAGAGAACCGCCAGAAGAA
GATACACCAGACGGAAGAACCGGATCTGCTAT CT GCAAGAGATCTT CAGCAACGAGAT GGCCAAGGT G
GACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCA
CCCCAT CT TCGGCAACAT CGTGGACGAGGT GGCCTACCACGAGAAGTACCCCACCATCTACCACCTGA
GAAAGAAACT GGTGGACAGCACCGACAAGGCCGACCTGCGGCTGAT CTAT CT GGCCCT GGCCCACAT G
AT CAAGTT CCGGGGCCACTT CCTGAT CGAGGGCGACCT GAACCCCGACAACAGCGACGTGGACAAGCT
GT TCAT CCAGCT GGTGCAGACCTACAACCAGCTGTT CGAGGAAAACCCCATCAACGCCAGCGGCGTGG
ACGCCAAGGCCATCCT GT CT GCCAGACT GAGCAAGAGCAGACGGCT GGAAAATCTGAT CGCCCAGCT G
CCCGGCGAGAAGAAGAATGGCCTGTTCGGCAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTT
CAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACC
TGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCC
GACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTC
TATGAT CAAGAGATACGACGAGCACCACCAGGACCT GACCCT GCTGAAAGCT CT CGTGCGGCAGCAGC
TGCCTGAGAAGTACAAAGAGAT TT TCTT CGACCAGAGCAAGAACGGCTACGCCGGCTACATT GACGGC
GGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGA
ACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCC
CCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTG
AAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGC
CAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCG
AGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAAC
CT GCCCAACGAGAAGGTGCT GCCCAAGCACAGCCTGCT GTACGAGTACTT CACCGT GTATAACGAGCT
GACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGG
CCAT CGTGGACCTGCT GT TCAAGACCAACCGGAAAGTGACCGTGAAGCAGCT GAAAGAGGACTACTT C
AAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGG
CACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACA

T T CT GGAAGATAT C GT GC T GAC CC T GACAC T GT T T GAGGACAGAGAGAT GAT CGAGGAAC
GGCT GAAA
ACCTAT GCCCACCT GT T CGACGACAAAGT GAT GAAGCAGC T GAAGCGGCGGAGATACACCGGCT GGGG

CAGGCT GAGCCGGAAGCT GAT CAACGGCAT CCGGGACAAGCAGT CCGGCAAGACAAT CCT GGAT T T CC

TGAAGTCCGACGGCTTCGCCAACAGAAACT T CAT GCAGCT GAT CCACGACGACAGCCT GACC T T TAAA
GAGGACAT CCAGAAAGCCCAGGT GT CCGGCCAGGGCGATAGCCT GCACGAGCACAT TGCCAATCTGGC
CGGCAGCCCCGCCAT TAAGAAGGGCAT CCT GCAGACAGT GAAGGT GGT GGACGAGC T CGT GAAAGT GA

T GGGCCGGCACAAGCCCGAGAACAT CGT GAT CGAAAT GGCCAGAGAGAAC CAGACCACCCAGAAGGGA
CAGAAGAACAGCCGCGAGAGAAT GAAGCGGAT CGAAGAGGGCAT CAAAGAGC T GGGCAGCCAGAT CC T
GAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATG
GGCGGGATAT GTACGT GGACCAGGAACT GGACAT CAACCGGC T GT CCGAC TACGAT GT GGACCACAT
C
GT GCCT CAGAGC T T TCT GAAGGACGACT CCAT CGACAACAAGGT GC T
GACCAGAAGCGACAAGGCCCG
GGGCAAGAGCGACAACGT GCCC T CCGAAGAGGT CGT GAAGAAGAT GAAGAAC TACT GGCGGCAGCT GC

T GAACGCCAAGC T GAT TACCCAGAGAAAGT T C GACAAT CT GACCAAGGCC GAGAGAGGCGGC CT
GAGC
GAAC T GGATAAGGCCGGC T T CAT CAAGAGACAGC T GGT GGAAACCCGGCAGAT CACAAAGCACGT
GGC
ACAGAT CC T GGACT CCCGGAT GAACACTAAGTAC GACGAGAAT GACAAGC T GAT CCGGGAAGT
GAAAG
T GAT CACCCT GAAGT CCAAGCT GGT GT CCGAT TTCCGGAAGGAT TTCCAGTT TTACAAAGTGCGCGAG

AT CAACAACTACCACCACGCCCACGACGCC TACC T GAACGCCGT CGT GGGAACCGCCC T GAT CAAAAA
GTACCC TAAGCT GGAAAGCGAGT T CGT GTACGGCGACTACAAGGT GTACGACGT GCGGAAGAT GAT CG

CCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACT TCT T CTACAGCAACAT CAT GAACT T T
TTCAAGACCGAGAT TACCCT GGCCAACGGCGAGAT CCGGAAGCGGCCT CT GAT CGAGACAAACGGCGA
AACCGGGGAGAT CGT GT GGGATAAGGGCCGGGAT TT T GCCACCGT GCGGAAAGT GC T GAGCAT
GCCCC
AAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCT T CAGCAAAGAGT CTAT CC T GCCCAAG
AGGAACAGCGATAAGC T GAT CGCCAGAAAGAAGGAC T GGGAC CC TAAGAAGTAC GGCGGC T T
CGACAG
CCCCACCGTGGCCTAT T C T GT GCT GGT GGT GGCCAAAGT GGAAAAGGGCAAGT CCAAGAAAC T
GAAGA
GT GT GAAAGAGC T GCT GGGGAT CACCAT CAT GGAAAGAAGCAGC T T CGAGAAGAAT CCCAT
CGACT T T
CT GGAAGCCAAGGGCTACAAAGAAGT GAAAAAGGACCT GAT CAT CAAGCT GCCTAAGTAC T CCC T GT
T
CGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGG
CCCT GCCC T CCAAATAT GT GAACT T CCT GTACCT GGCCAGCCAC TAT GAGAAGC T GAAGGGC T
CCCCC
GAGGAT AAT GAGCAGAAACAGC T GT T T GT GGAACAGCACAAGCACTACCT GGAC GAGAT CAT
CGAGCA
GAT CAGCGAGT T CT CCAAGAGAGT GAT CCT GGCCGACGCTAAT C T GGACAAAGT GC T GT CCGCC
TACA
ACAAGCACCGGGATAAGCCCAT CAGAGAGCAGGCCGAGAATAT CAT CCACCT GT TTACCCTGACCAAT
CT GGGAGCCCCT GCCGCC T T CAAGTACT TTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAA
AGAGGT GC T GGACGCCACCC T GAT CCACCAGAGCAT CACCGGCC T GTACGAGACACGGAT CGACCT
GT
CT CAGC T GGGAGGCGACAGCGC T GGAGGAGGT GGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTAGC
GGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGGATCCGGACGGGCTGACGCATTGGACGATT T T GA
T C T GGATAT GCT GGGAAGT GACGCCC T CGAT GAT TT TGACCT T GACAT GC T T GGT T
CGGAT GCCCT T G
AT GACT TI GACC T CGACAT GCT CGGCAGT GACGCCC T T GAT GAT TI CGACCT GGACAT GC T
GAT TAAC
TGTACATAAACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACT
CCAG TGCCCACCAGCC TTGTCC TAATAAAAT TAAGT TGCATCAT TT TG TC TGAC TAGG TG TCCT
TC TA
TAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCT
GC GGGG TC TATTGGGAAC CAAGCTGGAG TGCAGTGGCACAATCT TGGC TCAC TGCAATCTCCGCCTC C
TGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCT
CAGC TAAT TT TTGT TT TT TTGGTAGAGACGGGGT TTCACCATAT TGGCCAGGCTGGTC TCCAAC TCC
T

AATC TCAGGTGATC TACC CACC TTGGCC TC CCAAAT TGC TGGGATTACAGGC GTGAAC CAC TGC
TCC C
TTCCCTGTCCTTGAAT TCTAACTATAACGGTCCTAAGGTAGCGAAGCTAGCT GCAAAGAT GGATAAAG
TTTTAAACAGAGAGGAAT CT TT GCAGCTAATGGACCTT CT AGGT CT TGAAAGGAGT GGGAAT T
GGCTC
CGGTGC CCGT CAGTGGGCAGAGCGCACATCGC CCACAG TC CC CGAGAAGT TGGGGGGAGGGGTCGGCA
AT TGAACCGG TG CC TAGAGAAGGTGGCGCGGGGTAAAC TGGGAAAG TGATGT CG TG TAC TGGC T
CCGC
C T T T T T CC CGAGGG TGGGGGAGAACCGTATATAAGTGCAG TAGT CGCCGTGAACGT TCTTTT
TCGCAA
CGGGTT TGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGT TCCCGCGGGCCTGGCCTCTT TACGGGT T
ATGGCCCT TGCGTGCCTTGAAT TACT TCCACC TGGC TGCAGTACGTGAT T CT TGATCCCGAGCT TCGG
GT TGGAAGTGGGTGGGAGAGTTCGAGGCCT TGCGCT TAAGGAGC CC C T TCGCCTCGTGCT TGAGTTGA
GGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCT TCGCGC CTGT CT CGCTGCT T
T CGATAAGTC TC TAGC CAT T TAAAAT TT TTGATGACCTGCTGCGACGCTTTTTT TCTGGCAAGATAGT
CT TG TAAATG CGGGCCAAGATC TGCACAC TGG TAT T TCGG T T T T
TGGGGCCGCGGGCGGCGACGGGGC
CCGTGCGT CC CAGCGCACATGT TCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGG
GGTAGT CT CAAGCTGGCCGGCC TGCT CTGGTGCC TGGCCT CGCGCCGCCGTGTATCGCCCCGCCCTGG
GCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGC
AGGGAGCT CAAAATGAAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAA
GGGCCT T T CCGT CC TCAGCCGT CGCT TCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCT C
GAT TAG T T C T CGAGC T TT TGGAGTACGTCGTC TT TAGGTTGGGGGGAGGGGT TT
TATGCGATGGAGT T
TC CC CACAC TGAGTGGGTGGAGAC TGAAGT TAGGCCAGCT TGGCACTTGATGTAAT TCTCCT TGGAAT
TTGCCCTT TT TGAGTT TGGATC T TGGT T CAT T CT CAAGCC T CAGACAGTGGT TCAAAGT T T T
T T TCTT
CCA T TTCAGG TG TCGTGACGTACGGC CACCATGGC T TCAAAC TT TAC TCAGT TCGTGC
TCGTGGACAA
TGGTGGGACAGGGGATGTGACAGTGGC TCC TTC TAATT TC GC TAATGGGG TGGCAGAG TGGATCAGC T
CCAAC TCACGGAGC CAGGCC TACAAGGTGACATGCAGC GTCAGGCAGTC TAG TGCC CAGAAGAGAAAG
TATACCATCAAGGTGGAGGTCCCCAAAGTGGCTACCCAGACAGTGGGCGGAGTCGAACTGCCTGTCGC
CGC T TGGAGGTCC TACC TGAACATGGAGC TCAC TATCCCAAT TT TCGC TACCAATTC TGAC
TGTGAAC
TCATCG TGAAGGCAATGCAGGGGC TC C TCAAAGACGGTAATC C TATCC C T TC CGCCATCGCC GC
TAAC
TCAGGTATCTACAGCGCTGGAGGAGGTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTAGCGGACC
TAAGAAAAAGAGGAAGGTGGCGGC CGC TGGATCC CC TTCAGGGCAGATCAGCAACCAGGC CC TGGC TC
TGGCCCCTAGCTCCGCTCCAGTGCTGGCCCAGACTATGGTGCCCTCTAGTGCTATGGTGCCTCTGGCC
CAGCCACCTGCTCCAGCCCCTGTGCTGACCCCAGGACCACCCCAGTCACTGAGCGCTCCAGTGCCCAA
GTC TACACAGGC CGGC GAGGGGAC TC TGAG TGAAGC TC TGC TGCAC C TGCAG TTCGAC GC
TGATGAGG
AC C TGGGAGC TC TGC TGGGGAACAGCAC CGATCC CGGAGTGT TCACAGATC TGGCC TC CG
TGGACAAC
TC TGAG TT TCAGCAGC TGC TGAATCAGGGC GTGTCCATGTC TCATAGTACAGCC GAAC CAATGC TGAT

GGAGTACCCCGAAGCCATTACCCGGCTGGTGACCGGCAGCCAGCGGCCCCCCGACCCCGCTCCAACTC
CC C TGGGAAC CAGC GGCC TGCC TAATGGGC TG TC CGGAGATGAAGAC T TC TCAAGCATCGC
TGATATG
GACTTTAGTGCCCTGCTGTCACAGATTTCCTCTAGTGGGCAGGGAGGAGGTGGAAGCGGCTTCAGCGT
GGACACCAGTGCCCTGCTGGACCTGTTCAGCCCCTCGGTGACCGTGCCCGACATGAGCCTGCCTGACC
TTGACAGCAGCC TGGC CAGTATCCAAGAGC TC C TGTC TCC CCAGGAGC CC CC CAGGCC TC CC
GAGGCA
GAGAACAGCAGC CC GGAT TCAGGGAAGCAGC TGG TGCAC TACACAGCGCAGC CGC TGT TC C TGC
TGGA
CCCCGGCTCCGTGGACACCGGGAGCAACGACCTGCCGGTGCTGTTTGAGCTGGGAGAGGGCTCCTACT
TC TC CGAAGGGGAC GGC T TC GC CGAGGACC CCAC CATC TC CC TGC TGACAGGC TCGGAGC C
TCC CAAA
GCCAAGGACCCCACTGTCTCCTGTACATAAACGGGT GGCAT C CC T GT GAC CC C T CC CCAGT GCC
TC T C
CTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTT

GTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTG
GGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGG
CTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATT
CCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGG
CCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTA
CAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTGAATTCTAACTATAACGGTCCTAAGGTAGCGAAGG
TACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGA
CTGGAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTT
AGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCT
TGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGA
CCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGCATCTAGAATTAATTCCGTGTATTCTATAGTGTCAC
CTAAATCGTATGTGTATGATACATAAGGTTATGTATTAATTGTAGCCGCGTTCTAACGACAATATGTA
CAAGCCTAATTGTGTAGCATCTGGCTTACTGAAGCAGACCCTATCATCTCTCTCGTAAACTGCCGTCA
GAGTCGGTTTGGTTGGACGAACCTTCTGAGTTTCTGGTAACGCCGTCCCGCACCCGGAAATGGTCAGC
GAACCAATCAGCAGGGTCATCGCTAGCCAGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGG
CGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACT
TCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGC
GCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTG
CTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAATGGTGCACTCTCAGTACAATCTGCTCTGAT
GCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCT
CCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGT
CATCACCGAAACGCGCG
RightITR = first underlined and bold sequence Packaging Signal = first underlined sequence RRE = first bold sequence U6 = second underlined sequence CMV = second bold sequence dCas9VP64 = third underlined sequence HGHpA = third bold sequence EFla = fourth underlined sequence MPH = fourth bold sequence HGHpA = fifth underlined sequence LeftITR = second underlined and bold sequence SEQ ID NO:10 AAV-dCas9VP64 LeftITR-EFla-dCas9VP64-FpA-RightITR
GTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAA
AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAA

CIGGCTICAGCAGAGCGCAGATACCAAATACTGICCTICTAGTGTAGCCGTAGTTAGGCCACCACTIC
AAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCIGTTACCAGTGGCTGCTGCCAGTGG
CGATAAGTCGTGICTTACCGGGITGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGICGGGCT
GAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAG
CGTGAGCTATGAGAAAGCGCCACGCT TCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAG
GGICGGAACAGGAGAGCGCACGAGGGAGCTICCAGGGGGAAACGCCIGGTATCTITATAGTCCTGICG
GGITTCGCCACCICTGACTTGAGCGTCGATTITTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAA
AACGCCAGCAACGCGGCCTT TT TACGGT TCCIGGCCIT TTGCTGGCCT TT TGCTCACATGT CC TGCAG

GCAGC TGC GC GC TC GC TC GC TCAC TGAGGC CGCC CGGGCAAAGC CC GGGC GTCGGGCGAC C T
TTGGTC
GC CC GGCC TCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGC
GGCCGCACGCGTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTT
GGGGGGAGGGGICGGCAATTGAACCGGIGCCTAGAGAAGGIGGCGCGGGGTAAACTGGGAAAGTGATG
TCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTG
AACGTTCT TT TTCGCAACGGGT TTGCCGCCAGAACACAGGAATT C G C CAC CATGAAAAGGCCGGCGGC
CACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGGACAAGAAGTACAGCATCGGCC TGGCCATCGGCA
CCAACTCTGTGGGC TGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTG
GGCAACACCGACCGGCACAGCATCAAGAAGAACC TGATCGGAGC CC TGCTGTTCGACAGCGGCGAAAC
AGCC GAGGCCAC CC GGC TGAAGAGAACC GC CAGAAGAAGATACACCAGAC GGAAGAAC CGGATC TGC T

ATCTGCAAGAGATC TTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTC TTCCACAGAC TGGAAGAG
TCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGT
GGCC TACCACGAGAAGTACCCCACCATC TACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGG
CC GACC TGCGGC TGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCAC TTCC TGATCGAG
GGCGAC C TGAAC CC CGACAACAGC GACG TGGACAAGC TGT TCATCCAGC TGG TGCAGACC
TACAACCA
GC TG TTCGAGGAAAAC CC CATCAACGCCAGCGGC GTGGAC GC CAAGGC CATC C TGTC TGC CAGAC
TGA
GCAAGAGCAGACGGCTGGAAAATC TGATCGCCCAGC TGCCCGGCGAGAAGAAGAATGGCC TGTTCGGC
AACC TGAT TGCC C TGAGC C TGGGC C TGACC CC CAAC TTCAAGAGCAAC
TTCGACCTGGCCGAGGATGC
CAAACTGCAGCTGAGCAAGGACACCTACGACGACGACC TGGACAACCTGC TGGCCCAGATCGGCGACC
AG TACGCC GACC TG TT TC TGGCCGCCAAGAACCTGTCCGACGCCATCC TGCTGAGCGACATCCTGAGA
GTGAACAC CGAGATCACCAAGGCC CC CC TGAGCGCC TC TATGATCAAGAGATACGACGAGCACCACCA
GGACCTGACCCTGC TGAAAGCTCTCGTGCGGCAGCAGC TGCC TGAGAAGTACAAAGAGAT TT TC TTCG
AC CAGAGCAAGAAC GGC TAC GC CGGC TACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTC
ATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAAC TGCTCGTGAAGC TGAACAGAGAGGACC T
GC TGCGGAAGCAGC GGAC C T TC GACAAC GGCAGCATCC CC CACCAGATCCAC C TGGGAGAGC
TGCACG
CCATTC TGCGGC GGCAGGAAGATT TT TACC CATTCC TGAAGGACAACCGGGAAAAGATCGAGAAGATC
CTGACC TTCC GCATCC CC TAC TAC GTGGGC CC TC TGGCCAGGGGAAACAGCAGATTCGCC TGGATGAC
CAGAAAGAGC GAGGAAAC CATCAC CC CC TGGAAC TTCGAGGAAG TGGTGGACAAGGGC GC TTCCGCCC
AGAGCTTCATCGAGCGGATGACCAAC TTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCAC
AGCC TGCTGTACGAGTAC TTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAAT
GAGAAAGC CC GC C T TC C TGAGC GGCGAGCAGAAAAAGGCCATCG TGGACC TGCTGTTCAAGACCAACC
GGAAAGTGACCGTGAAGCAGCTGAAAGAGGAC TACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAA
ATCTCCGGCGTGGAAGATCGGTTCAACGCC TC CC TGGGCACATACCACGATC TGCTGAAAATTATCAA
GGACAAGGAC TTCC TGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGC TGAC CC TGACAC
TG TT TGAGGACAGAGAGATGATCGAGGAAC GGC TGAAAAC C TATGC CCAC C TGT TC
GACGACAAAGTG

ATGAAGCAGC TGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCAT
CC GGGACAAGCAGTCC GGCAAGACAATC C TGGAT TTCC TGAAGTCCGACGGC TTCGCCAACAGAAAC T
TCATGCAGCTGATCCACGACGACAGCCTGACC TT TAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGC
CAGGGCGATAGCCTGCACGAGCACAT TGCCAATC TGGC CGGCAGCC CC GC CATTAAGAAGGGCATCC T
GCAGACAGTGAAGGTGGTGGACGAGC TCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGA
TCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGG
ATCGAAGAGGGCATCAAAGAGC TGGGCAGC CAGATC C TGAAAGAACAC CC CG TGGAAAACAC CCAGC T
GCAGAACGAGAAGC TGTACC TGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGG
ACATCAACCGGC TGTCCGAC TACGATGTGGACCACATCGTGCCTCAGAGC TT TC TGAAGGACGACTCC
ATCGACAACAAGGTGC TGAC CAGAAGCGACAAGGCC CGGGGCAAGAGC GACAAC GTGC CC TC CGAAGA
GGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGT
TCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAAC TGGATAAGGCCGGC TTCATCAAGAGA
CAGC TGGTGGAAAC CC GGCAGATCACAAAGCACG TGGCACAGATCC TGGACTCCCGGATGAACACTAA
GTACGACGAGAATGACAAGC TGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCG
AT TTCC GGAAGGAT TTCCAG TT TTACAAAG TGCGCGAGATCAACAAC TAC CACCAC GC CCAC GACGC
C
TACC TGAACGCC GTCG TGGGAACC GC CC TGATCAAAAAGTAC CC TAAGC TGGAAAGCGAG TTCG
TGTA
CGGC GAC TACAAGG TG TACGAC GTGC GGAAGATGATCGCCAAGAGC GAGCAGGAAATC GGCAAGGC TA
CC GC CAAG TAC T TC TTCTACAGCAACATCATGAACT TT TTCAAGACCGAGAT TACCCTGGCCAACGGC
GAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCG
GGAT TT TGCCACCGTGCGGAAAGTGC TGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGC
AGACAGGCGGCT TCAGCAAAGAGTCTATCC TGCCCAAGAGGAACAGCGATAAGC TGATCGCCAGAAAG
AAGGAC TGGGAC CC TAAGAAGTACGGCGGC TTCGACAGCC CCAC CG TGGC C TAT TC
TGTGCTGGTGGT
GGCCAAAGTGGAAAAGGGCAAGTCCAAGAAAC TGAAGAGTGTGAAAGAGC TGCTGGGGATCACCATCA
TGGAAAGAAGCAGC TTCGAGAAGAATCCCATCGACT TTCTGGAAGCCAAGGGCTACAAAGAAGTGAAA
AAGGACCTGATCATCAAGCTGCCTAAGTAC TC CC TGTTCGAGCTGGAAAACGGCCGGAAGAGAATGC T
GGCC TC TGCC GGCGAAC TGCAGAAGGGAAACGAAC TGGCC C TGC CC TCCAAATATGTGAACT TCCTGT

AC C TGGCCAGCCAC TATGAGAAGC TGAAGGGC TC CC CC GAGGATAATGAGCAGAAACAGC TG TT
TGTG
GAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCC T
GGCCGACGCTAATC TGGACAAAGTGC TGTCCGCC TACAACAAGCACCGGGATAAGCCCATCAGAGAGC
AGGCCGAGAATATCATCCACCTGT TTAC CC TGAC CAATC TGGGAGC CC C TGC CGCC TTCAAG TAC
TT T
GACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGC TGGACGCCAC CC TGATCCACCA
GAGCATCACCGGCC TGTACGAGACACGGATCGACCTGTCTCAGC TGGGAGGC GACAGC GC TGGAGGAG
GTGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGTAGCGGACCTAAGAAAAAGAGGAAGGTGGCGGCC
GC TGGATC CGGACGGGC TGACGCATTGGAC GATT TTGATC TGGATATGC TGGGAAG TGAC GC CC
TCGA
.. TGAT TT TGACCT TGACATGC TTGGTTCGGATGCCCT TGATGACT TTGACC TCGACATGCTCGGCAGTG
AC GC CC TTGATGAT TTCGACCTGGACATGC TGTAAC T C GAGCAATAAAGAAT CGTT TGIGTTATGT
TT
CAACGTGT TTAT TT TTCAAT TGCAGCGGACCGAGCGGCCGCAGGAACCCCTAGTGATGGAGTTGGCCA
CTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTT
GCCCGGGCGGCC TCAGTGAGCGAGCGAGCGCGCAGC TGCC TGCAGGGGCGCC T GAT GCGGTATTTTCT
CCITACGCATCTGTGCGGTATTICACACCGCATACGTCAAAGCAACCATAGTACGCGCCCIGTAGCGG
CGCATTAAGCGCGGCGGGIGIGGIGGITACGCGCAGCGTGACCGCTACACTIGCCAGCGCCCTAGCGC
CCGCTCCITICGCTTICTICCCTICCITTCTCGCCACGTTCGCCGGCTTICCCCGTCAAGCTCTAAAT
CGGGGGCTCCCITTAGGGITCCGATTTAGTGCTITACGGCACCTCGACCCCAAAAAACTTGATTIGGG

TGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGT
TCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCTTTTGAT
TTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGC
GAATTTTAACAAAATATTAACGTTTACAATTTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCG
CATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCG
GCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATC
ACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAA
TGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTC
TAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAA
AAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTC
CTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTG
GGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCC
AATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGC
AACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCAT
CTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGC
CAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATC
ATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACC
ACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTC
CCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTC
CGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCA
CTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGA
TGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAG
TTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATC
CTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCC
RightITR = first underlined and bold sequence EFla = first underlined sequence dCas9VP64 = bold sequence FpA = second underlined sequence LeftITR = second underlined and bold sequence SEQ ID NO: 11 AAV-MPH
LeftITR-CMV-MPH-HGHpA-U6-0M-RightITR
AGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCA
CCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTT
CAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACT
CTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG
TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGG
GGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC

TATGAGAAAGCGCCACGCTICCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGICGGA
ACAGGAGAGCGCACGAGGGAGCTICCAGGGGGAAACGCCIGGTATCTITATAGTCCTGICGGGITTCG
CCACCICTGACTTGAGCGTCGATTITTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCA
GCAACGCGGCCT TT TTACGGITCCIGGCCT TT TGCTGGCCTT TTGCTCACATGT CC TGCAGGCAGC TG
CGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACC TT TGGTCGCCCGGC
C TCAGTGAGC GAGC GAGC GC GCAGAGAGGGAG TGGC CAAC TCCATCAC TAGGGGTTCC TGCGGCCGCA

CGCGTGGAGCTAGT TATTAATAGTAATCAATTACGGGGTCAT TAGT TCATAGCCCATATATGGAGTTC
CGCGTTACATAACTTACGGTAAATGGCCCGCCIGGCTGACCGCCCAACGACCCCCGCCCATTGACGTC
AATAATGACGTATGITCCCATAGTAACGTCAATAGGGACTITCCATTGACGTCAATGGGIGGAGTATT
TACGGTAAACTGCCCACTIGGCAGTACATCAAGIGTATCATATGCCAAGTACGCCCCCTATTGACGTC
AATGACGGTAAATGGCCCGCCIGGCATTATGCCCAGTACATGACCTTATGGGACTITCCTACTIGGCA
GTACATCTACGTAT TAGTCATCGCTATTACCATGGTGATGCGGT TT TGGCAGTACATCAATGGGCGTG
GATAGCGGITTGACTCACGGGGATTICCAAGICTCCACCCCATTGACGTCAATGGGAGITTGITTTGC
ACCAAAATCAACGGGACT TTCCAAAATGICGTAACAACTCCGCCCCAT TGACGCAAATGGGCGGTAGG
CGTGTACGGIGGGAGGICTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCA
TCCACGCTGITTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGATTCGAATCCCGGC
CGGGAACGGIGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGICTA
TAGGCCCACAAAAAATGCTT TCTICT TT TAATATACTT TT =GT TTATCT TATT TCTAATACTT TCCC
TAATCTCTTICTTICAGGGCAATAATGATACAATGTATCATGCCICITTGCACCATTCTAAAGAATAA
CAGTGATAAT TTCTGGGT TAAGGCAATAGCAATATT TCTGCATATAAATATT TCTGCATATAAATTGT
AACTGATGTAAGAGGITTCATATTGCTAATAGCAGCTACAATCCAGCTACCATTCTGCTITTATITTA
TGGT TGGGATAAGGCTGGAT TATTCTGAGTCCAAGCTAGGCCCT TT TGCTAATCATGT TCATACCTCT
TATCTICCTCCCACAGCTCCIGGGCAACGTGCTGGICTGIGTGCTGGCCCATCACTITGGCAAAGAAT
TGGGAT TCGAACAT CGAT TGAATT CAC CATGGC T TCAAAC TT TAC TCAGT TC GTGC
TCGTGGACAATG
GTGGGACAGGGGATGTGACAGTGGCTCC TTC TAATT TC GC TAATGGGGTGGCAGAGTGGATCAGCTCC
AACTCACGGAGCCAGGCC TACAAGGTGACATGCAGC GTCAGGCAGTC TAG TGCC CAGAAGAGAAAGTA
TACCATCAAGGTGGAGGTCCCCAAAGTGGC TACCCAGACAGTGGGCGGAGTCGAAC TGCC TGTCGCCG
C T TGGAGG TC C TAC C TGAACATGGAGC TCAC TATCC CAAT TT TC GC
TACCAATTCTGACTGTGAACTC
ATCGTGAAGGCAATGCAGGGGC TC C TCAAAGACGGTAATC C TATCC C T TC CGCCATCGCC GC
TAACTC
AGGTATC TACAGCGC TGGAGGAGG TGGAAGCGGAGGAGGAGGAAGC GGAGGAGGAGGTAGCGGACC TA
AGAAAAAGAGGAAGGTGGCGGC CGC TGGATCC CC TTCAGGGCAGATCAGCAACCAGGC CC TGGC TCTG
GCCCCTAGCTCCGC TCCAGTGC TGGCCCAGAC TATGGTGCCC TC TAGTGC TATGGTGCCTCTGGCCCA
GCCACC TGCTCCAGCCCC TGTGCTGACCCCAGGACCACCCCAGTCACTGAGCGC TCCAGTGCCCAAGT
CTACACAGGCCGGCGAGGGGAC TC TGAGTGAAGC TC TGC TGCAC C TGCAG TTCGAC GC TGATGAGGAC
CTGGGAGC TC TGCTGGGGAACAGCACCGATCCCGGAGTGTTCACAGATCTGGCC TCCGTGGACAACTC
TGAG TT TCAGCAGC TGCTGAATCAGGGCGTGTCCATGTCTCATAGTACAGCCGAACCAATGC TGATGG
AGTACCCCGAAGCCATTACCCGGC TGGTGACCGGCAGCCAGCGGCCCCCCGACCCCGC TCCAAC TCCC
CTGGGAACCAGCGGCC TGCC TAATGGGC TGTCCGGAGATGAAGACTTC TCAAGCATCGCTGATATGGA
CTTTAGTGCCCTGC TGTCACAGATTTCC TC TAGTGGGCAGGGAGGAGGTGGAAGCGGC TTCAGCGTGG
ACACCAGTGCCC TGCTGGACCTGTTCAGCCCC TCGGTGACCGTGCCCGACATGAGCCTGCCTGACCTT
GACAGCAGCC TGGCCAGTATCCAAGAGC TC C TGTC TCC CCAGGAGC CC CC CAGGCC TC CC
GAGGCAGA
GAACAGCAGC CC GGAT TCAGGGAAGCAGC TGG TGCAC TACACAGCGCAGC CGC TGT TC C TGC
TGGACC
CCGGCTCCGTGGACACCGGGAGCAACGACC TGCCGGTGCTGTTTGAGC TGGGAGAGGGCTCC TACTTC

TC CGAAGGGGAC GGC T TC GC CGAGGACC CCAC CATC TC CC TGC TGACAGGC TCGGAGC C TCC
CAAAGC
CAAGGACCCCACTGTCTCCTGACCTCGAGCAGCGCT GCTCGAGAGATCTACGGGIGGCATCCCIGTGA
CCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAA
AATTAAGTTGCATCATITTGICTGACTAGGIGTCCTICTATAATATTATGGGGIGGAGGGGGGIGGTA
TGGAGCAAGGGGCAAGTIGGGAAGACAACCIGTAGGGCCTGCGGGGICTATTGGGAACCAAGCTGGAG
TGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGC
CTCCCGAGTTGT TGGGAT TCCAGGCATGCATGACCAGGCTCAGCTAAT TT TTGT TT TT TTGGTAGAGA
CGGGGITTCACCATATTGGCCAGGCTGGICTCCAACTCCTAATCTCAGGTGATCTACCCACCTIGGCC
TCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTICCCTGICCTICTGATTTIGTAGGTAA
CCACGTGCGGACCTAGGGATAACAGGGTAAT GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATA
TACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAA
AATACG TGAC GTAGAAAG TAATAATT TC TTGGGTAG TT TGCAGT TT TAAAAT TATG TT
TTAAAATGGA
C TATCATATGC T TACC GTAAC T TGAAAG TATT TC GATT TC TTGGC T TTATATATC T TG
TGGAAAGGAC
GAAACACCGANNOMMOMMONGITTTAGAGCTAGGCCAACATGAGGATCACCCATGTCTG
CAGGGCCTAGCAAGTTAAAATAAGGCTAGTCCGT TATCAACT TGGCCAACATGAGGATCACCCATGTC
TGCAGGGCCAAGTGGCACCGAGTCGGTGCT TT TT TTGGATCCTGTTGACAAT TAATCATCGGCATAGT
ATATCGGCATAGTATAATACGACAAGGTGAGGAACTAAACCATGGCCAAGTTGACCAGTGCCGTTCCG
GTGCTCACCGCGCGCGACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGA
CTTCGTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGCGGTCCAGG
ACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCCTGGACGAGCTGTACGCCGAG
TGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCCTCCGGGCCGGCCATGACCGAGATCGGCGAGCA
GCCGTGGGGGCGGGAGTTCGCCCTGCGCGACCCGGCCGGCAACTGCGTGCACTICGTGGCCGAGGAGC
AGGACTGATAGGGATAACAGGGTAAT TAACTATAACGGICCTAAGGTAGCGAAGGACCGAGCGGCCGC
AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGA
CCAAAGGTCGCCCGACGCCCGGGC TT TGCCCGGGCGGCC TCAGTGAGCGAGCGAGCGCGCAGC TGCC T
GCAGGGGCGCCTGATGCGGTATTTICTCCITACGCATCTGTGCGGTATTICACACCGCATACGTCAAA
GCAACCATAGTACGCGCCCIGTAGCGGCGCATTAAGCGCGGCGGGIGTGGIGGITACGCGCAGCGTGA
CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTC
GCCGGCTITCCCCGTCAAGCTCTAAATCGGGGGCTCCCITTAGGGITCCGATTTAGTGCTITACGGCA
CCTCGACCCCAAAAAACTTGATTIGGGTGATGGITCACGTAGTGGGCCATCGCCCTGATAGACGGITT
TTCGCCCITTGACGTIGGAGTCCACGTTCTITAATAGTGGACTCTIGTTCCAAACTGGAACAACACTC
AACCCTATCTCGGGCTATTCTITTGATTTATAAGGGATITTGCCGATTTCGGCCTATTGGITAAAAAA
TGAGCTGATT TAACAAAAAT TTAACGCGAATT TTAACAAAATAT TAACGT TTACAATT TTATGGTGCA
CICTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGAC
GCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTG
CATGIGICAGAGGT TT TCACCGTCATCACCGAAACGCGCGAGACGAAAGGGCCTCGTGATACGCCTAT
TT TTATAGGT TAATGICATGATAATAATGGIT TCTTAGACGTCAGGIGGCACTT TTCGGGGAAATGTG
CGCGGAACCCCTATTTGITTATTITTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACC
CTGATAAATGCTICAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTICCGTGICGCCCITA
TTCCCITTITTGCGGCATITTGCCTICCTGITITTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGAT
GCTGAAGATCAGTIGGGIGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCITGA
GAGTITTCGCCCCGAAGAACGTITTCCAATGATGAGCACTITTAAAGTICTGCTATGIGGCGCGGTAT
TATCCCGTATTGACGCCGGGCAAGAGCAACTCGGICGCCGCATACACTATTCTCAGAATGACTIGGIT

GAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGC
CATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAA
CCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAA
GCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATT
AACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTG
CAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAG
CGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTA
CACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGA
TTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTT
TAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTT
TTCGTTCCACTGAGCGTCAGACCCCGTAGAAA
RightITR = first underlined and bold sequence CMV = first underlined sequence MPH = first bold sequence HGHpA = second underlined sequence U6 = second bold sequence LeftITR = second underlined and bold sequence SEQ ID NO : 1 2 AAV-dCas01-MPH-sgRNA
LeftITR-CBh-dCas(1)1-P2A-MPH-FpA-U6-KANN-RightITR
AGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCA
CCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTT
CAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACT
CTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAG
TCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGG
GGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGC
TATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGA
ACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCG
CCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCA
GCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTCCTGCAGGCAGCTG
CGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGC
CTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGCGGCCGCA
CGCGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGAC
GTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGT
ATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGAC
GTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTG
GCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTC
TCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCG

ATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGC
GAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGC
GGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGACGCTGCCTTCG
CCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCAC
AGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCTGAGCAAGAGGTAAGGGTTTAAG
GGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGGAGCACCTGTCCGGAGAATTCGCCACCAT
GAAAAGGC CGGC GGCCAC GAAAAAGGCC GGCCAGGCAAAAAAGAAAAAGGCC GATACC CC CACAC TG T
TCACCCAATTCC TCAGACACCACC TC CC CGGC CAAAGATT TAGAAAGGACAT TC TGAAGCAAGCCGGA
AGAATCCTCGCTAATAAGGGAGAGGACGCCACAATTGCCTTTCTGAGAGGCAAATCCGAGGAGAGCCC
TC CC GAC T TC CAAC CC CC CG TGAAGTGC CC CATCATCGC T TGCAGCAGAC C TC
TGACAGAATGGCCCA
TC TATCAAGCCAGCGTGGCTATCCAAGGCTACGTCTACGGCCAGTC TC TGGCCGAATTTGAGGCCAGC
GACC CC GGC TGT TC CAAGGATGGAC TCC TC GGATGG TT TGACAAGACC GGCG TC
TGCACCGATTATTT
CAGCGTGCAAGGAC TGAACC TCAT TT TC CAGAAC GC TAGGAAGAGGTATATCGGCGTGCAGACCAAGG
TGACCAATAGAAACGAAAAGAGGCACAAAAAGCTGAAGAGGATCAACGCCAAGAGAATCGCTGAAGGA
C TGC CC GAGC TGACCTCCGACGAGCCCGAGAGCGCTCTGGATGAAACCGGCCATCTGATCGACCCTCC
CGGACTGAACACAAACATCTAC TGC TAC CAGCAAGTGAGC CC TAAGCC TC TGGC TC TCAGCGAGGTGA
ATCAGC TGCC CACC GC C TAC GC TGGATACAGCACCTCCGGAGATGATCCCATCCAGCCCATGGTGACC
AAAGATAGAC TGAGCATC TC CAAAGGCCAGCC CGGATATATC CC CGAGCACCAGAGGGC TC TGC TGAG
CCAAAAGAAGCATAGAAGGATGAGAGGC TACGGACTGAAGGC TAGGGC TC TGCTCGTGATCGTGAGGA
TTCAAGATGACTGGGCCGTCATCGATCTGAGGTC TC TGCTGAGGAACGCTTACTGGAGGAGGATCGTC
CAGACAAAGGAGCCCTCCACAATCACCAAGCTGC TCAAGC TCGTGACCGGCGATCCCGTGCTGGACGC
CACCAGAATGGTCGCCAC C T TCAC C TATAAAC CC GGAATC GTGCAAGTGAGGAGCGC TAAATGTC TGA

AGAACAAGCAAGGCAGCAAGCTGTTCAGCGAAAGGTATCTGAACGAAACCGTGAGCGTGACCAGCATT
GC CC TC GGC TCCAACAATC TGG TC GC TGTGGCCACC TACAGAC TGG TCAACGGAAATACC CC
CGAAC T
GC TGCAGAGG TT TACAC TCC C TAGCCATC TGG TGAAGGAT TTCGAGAGGTACAAACAAGC TCACGATA

CAC TGGAGGAC TCCAT TCAGAAGACC GC CG TGGC TTC TC TGC CC CAAGGC CAGCAAAC CGAGAT
TAGA
ATGTGGTCCATGTACGGC TT TAGAGAGGCC CAAGAGAGGG TC TGTCAAGAGC TGGGAC TGGCCGACGG
ATCCATCCCTTGGAATGTGATGACCGCCACATCCACCATTCTGACAGATC TC TT TC TGGCCAGAGGAG
GAGACCCCAAGAAGTGCATGTTCACCAGCGAGCCCAAGAAGAAGAAGAAC TC CAAGCAAG TGC TC TAT
AAGATTAGAGATAGAGCTTGGGCCAAGATGTACAGAACAC TGCTGTCCAAAGAGACCAGAGAGGCTTG
GAATAAAGCTCTGTGGGGAC TGAAAAGGGGCAGC CC CGAC TATGCCAGAC TGTCCAAGAGGAAGGAAG
AGCTGGCTAGAAGATGCGTCAACTACACCATC TC CACC GC CGAGAAGAGGGC CCAG TG TGGAAGGAC C
AT TG TGGC CC TCGAAGATCTGAACATCGGC TTC T TC CACGGCAGAGGAAAACAAGAGC CC GGATGGG
T
GGGAC TGT TCACAAGAAAGAAGGAGAACAGATGGC TCATGCAAGCC C TCCACAAGGC T TT TC TGGAGC
TGGC TCATCATAGAGGC TAC CACG TCATCGAAGTCAAC CC CGCC TATACC TC CCAGACATGC CC
CGTG
TG TAGACATTGC GACC CC GACAATAGAGAC CAGCATAACAGAGAGGCC TTCCAC TGTATCGGATGTGG
CTTCAGAGGCAACGCTGACC TCGACGTGGCCACCCACAACATTGCTATGGTGGCCATCACCGGCGAAT
CC C TCAAAAGGGCCAGAGGC TCCGTGGC TTCCAAGACACC TCAACC TC TGGCCGCCGAGGGCAGT G GA
GAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCAGCCACCATGGCTTC
AAAC TT TAC TCAGT TC GTGC TCGTGGACAATGGTGGGACAGGGGATGTGACAGTGGCTCC TTCTAATT
TC GC TAATGGGGTGGCAGAGTGGATCAGCTCCAACTCACGGAGCCAGGCC TACAAGGTGACATGCAGC
GTCAGGCAGTC TAG TGCC CAGAAGAGAAAG TATACCATCAAGGTGGAGGTCC CCAAAG TGGC TACCCA
GACAGTGGGCGGAGTCGAAC TGCC TG TC GC CGC T TGGAGG TC C TAC C TGAACATGGAGC TCAC
TATC C

CAAT TT TC GC TACCAATTC TGAC TGTGAAC TCATCG TGAAGGCAATGCAGGGGC TC C TCAAAGACGG
T
AATCCTATCCCTTCCGCCATCGCCGCTAACTCAGGTATCTACAGCGCTGGAGGAGGTGGAAGCGGAGG
AGGAGGAAGC GGAGGAGGAGGTAGCGGACC TAAGAAAAAGAGGAAGGTGGCGGC CGC TGGATCC CC T T
CAGGGCAGATCAGCAACCAGGC CC TGGC TC TGGC CC C TAGC TCC GC TC CAGTGC TGGC CCAGAC
TATG
GTGCCCTCTAGTGCTATGGTGCCTCTGGCCCAGCCACCTGCTCCAGCCCCTGTGCTGACCCCAGGACC
AC CC CAGTCAC TGAGC GC TC CAGTGC CCAAGTC TACACAGGC CGGC GAGGGGAC TC TGAG
TGAAGC TC
TGC TGCAC C TGCAG TTCGAC GC TGATGAGGAC C TGGGAGC TC TGC TGGGGAACAGCAC CGATCC
CGGA
GTGT TCACAGATC TGGCC TC CG TGGACAAC TC TGAG TT TCAGCAGC TGC TGAATCAGGGC
GTGTCCAT
GTC TCATAGTACAGCC GAAC CAATGC TGATGGAG TACC CC GAAGCCAT TACC CGGC TGGTGACC
GGCA
GCCAGCGGCCCCCCGACCCCGCTCCAACTCCCCTGGGAACCAGCGGCCTGCCTAATGGGCTGTCCGGA
GATGAAGACTTCTCAAGCATCGCTGATATGGACTTTAGTGCCCTGCTGTCACAGATTTCCTCTAGTGG
GCAGGGAGGAGGTGGAAGCGGCTTCAGCGTGGACACCAGTGCCCTGCTGGACCTGTTCAGCCCCTCGG
TGACCGTGCCCGACATGAGCCTGCCTGACCTTGACAGCAGCCTGGCCAGTATCCAAGAGCTCCTGTCT
CC CCAGGAGC CC CC CAGGCC TC CC GAGGCAGAGAACAGCAGC CC GGAT TCAGGGAAGCAGC TGG
TGCA
CTACACAGCGCAGCCGCTGTTCCTGCTGGACCCCGGCTCCGTGGACACCGGGAGCAACGACCTGCCGG
TGCTGT TTGAGC TGGGAGAGGGCTCC TACT TC TCCGAAGGGGACGGCT TCGCCGAGGACCCCACCATC
TC CC TGC TGACAGGC TCGGAGC C TCC CAAAGC CAAGGACC CCAC TG TC TC C T GACC T
CGAGCAATAAA
GAATCGTT TGTGTTAT GT TTCAACGT GT TTAT TT TTCAAT TGCAGCGGACCTAGGGATAACAGGGTAA
T GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTG
GAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTT
GGGTAG TT TGCAGT TT TAAAAT TATG TT TTAAAATGGAC TATCATATGC T TACC GTAAC T
TGAAAGTA
TT TCGATT TC TTGGCT TTATATATCT TGTGGAAAGGACGGCCAACAT GAGGAT CAC CCAT GTCTGCAG
GGCC CACC GGGAGAGAT C T CAAAC GAT T GC T C GAT T AGT C GAGACAGAAGAGCONAMMONAM
OfiNONGCTCT TCAT TT TT TT TGGTACCTAGGGATAACAGGGTAATTAACTATAACGGICCTAAGGTAG
CGAAGGAC CGAGCGGC CGCAGGAACCCC TAGTGATGGAGT TGGCCACTCCCTCTCTGCGCGC TCGC TC
GC TCAC TGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCT TTGCCCGGGCGGCC TCAGTGAGCG
AGCGAGCGCGCAGC TGCC TGCAGGGGCGCC T GAT GCGGTATT TICTCCITACGCATCT GT GCGGTATT
TCACACCGCATACGTCAAAGCAACCATAGTACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTG
GT GGTTACGCGCAGCGTGACCGCTACACTT GCCAGCGCCCTAGCGCCCGCTCCT TTCGCT TI CT TCCC
TTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCC
GATT TAGT GCTT TACGGCACCTCGACCCCAAAAAACTT GATT TGGGTGAT GGTTCACGTAGT GGGCCA
TCGCCCTGATAGACGGTT TT TCGCCCTT TGACGT TGGAGTCCACGT TCTT TAATAGTGGACTCT TGT T
CCAAACTGGAACAACACTCAACCCTATCTCGGGCTATTCT TT TGAT TTATAAGGGATT TT GCCGATT T
CGGCCT AT TGGT TAAAAAAT GAGC T GAT TTAACAAAAATT TAAC GC GAAT TT
TAACAAAATATTAACG
TT TACAAT TT TATGGT GCACTCTCAGTACAATCT GCTCTGAT GCCGCATAGT TAAGCCAGCCCCGACA
CCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTG
TGACCGTCTCCGGGAGCT GCAT GT GTCAGAGGTT TTCACCGTCATCACCGAAACGCGCGAGACGAAAG
GGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAATAATGGTTTCTTAGACGTCAGGTGG
CACT TT TCGGGGAAAT GT GCGCGGAACCCCTATT TGTT TATT TT TCTAAATACATTCAAATATGTATC
CGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAA
CATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAAC
GCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCA
ACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTT

CTGCTATGIGGCGCGGIATTATCCCGTATTGACGCCGGGCAAGAGCAACTCGGICGCCGCATACACTA
TICTCAGAATGACTIGGITGAGTACTCACCAGICACAGAAAAGCATCTTACGGATGGCATGACAGTAA
GAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTICTGACAACGATC
GGAGGACCGAAGGAGCTAACCGCTITITTGCACAACATGGGGGATCATGTAACTCGCCITGATCGTIG
GGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCIGTAGCAATGGCAA
CAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTICCCGGCAACAATTAATAGACTGG
ATGGAGGCGGATAAAGITGCAGGACCACTICTGCGCTCGGCCCTICCGGCTGGCTGGITTATTGCTGA
TAAATCTGGAGCCGGIGAGCGIGGGICTCGCGGIATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT
CCCGTATCGTAGITATCTACACGACGGGGAGICAGGCAACTAIGGATGAACGAAATAGACAGATCGCT
GAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGAT
TGATTTAAAACTICATTITTAATTTAAAAGGATCTAGGIGAAGATCCITITTGATAATCTCATGACCA
AAATCCCITAACGTGAGITTICGTICCACTGAGCGICAGACCCCGTAGAAA
RightITR = first underlined and bold sequence CBh = first underlined sequence dCas01 = first bold sequence P2A = second underlined sequence MPH = second bold sequence FpA = third underlined sequence U6 = third bold sequence LeftITR = second underlined and bold sequence OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a mammal having a polycystic kidney disease (PKD), wherein said method comprises administering to said mammal nucleic acid encoding a polycystin-1 (PC-1) polypeptide or a variant of said PC-1 polypeptide, wherein said PC-1 polypeptide or said variant is expressed by kidney cells within said mammal.

2. The method of claim 1, wherein said nucleic acid encoding said PC-1 polypeptide or said variant is administered to said mammal in the form of a viral vector.

3. The method of claim 2, wherein said viral vector is a helper-dependent adenovirus (HDAd) vector.

4. The method of any one of claims 1-3, wherein said nucleic acid encoding said PC-1 polypeptide or said variant is operably linked to a promoter sequence.

5. The method of claim 4, wherein said promoter sequence is selected from the group consisting of a human elongation factor la (EF1a) promoter sequence, a chicken 0-actin hybrid (CBh) promoter sequence, a PKD1 promoter sequence, a PKD2 promoter sequence, a cytomegalovirus (CMV) promoter sequence, a Rous sarcoma virus (RSV) promoter sequence, an aquaporin 2 (AQP2) promoter sequence, a gamma-glutamyltransferase 1 (Ggtl) promoter sequence, and a Ksp-cadherin promoter sequence.

6. A method for treating a mammal having a polycystic kidney disease (PKD), wherein said method comprises administering to said mammal nucleic acid encoding a polycystin-2 (PC-2) polypeptide or a variant of said PC-2 polypeptide, wherein said PC-2 polypeptide or said variant is expressed by kidney cells within said mammal.

7. The method of claim 6, wherein said nucleic acid encoding said PC-2 polypeptide or said variant is administered to said mammal in the form of a viral vector.

8. The method of claim 7, wherein said viral vector is an adenovirus-associated virus (AAV) vector.

9. The method of any one of claims 6-8, wherein said nucleic acid encoding said PC-2 polypeptide or said variant is operably linked to a promoter sequence.

10. The method of claim 9, wherein said promoter sequence is selected from the group consisting of a EFla promoter sequence, a CBh promoter sequence, a PKD1 promoter sequence, a PKD2 promoter sequence, a CMV promoter sequence, a RSV promoter sequence, an AQP2 promoter sequence, a Ggtl promoter sequence, and a Ksp-cadherin promoter sequence.

11. A method for treating a mammal having a polycystic kidney disease (PKD), wherein said method comprises administering to said mammal:
(a) nucleic acid encoding a PC-1 polypeptide or a variant of said PC-1 polypeptide, wherein said PC-1 polypeptide or said variant is expressed by kidney cells within said mammal; and (b) nucleic acid encoding a PC-2 polypeptide or a variant of said PC-2 polypeptide, wherein said PC-2 polypeptide or said variant is expressed by kidney cells within said mammal.

12. The method of claim 11, wherein said nucleic acid encoding said PC-1 polypeptide or said variant is administered to said mammal in the form of a viral vector.

13. The method of claim 12, wherein said viral vector is a HDAd vector.

14. The method of any one of claims 11-13, wherein said nucleic acid encoding said PC-1 polypeptide or said variant is operably linked to a promoter sequence.

15. The method of claim 14, wherein said promoter sequence is selected from the group consisting of a EFla promoter sequence, a CBh promoter sequence, a PKD1 promoter sequence, a PKD2 promoter sequence, a CMV promoter sequence, a RSV promoter sequence, an AQP2 promoter sequence, a Ggtl promoter sequence, and a Ksp-cadherin promoter sequence.

16. The method of any one of claims 11-15, wherein said nucleic acid encoding said PC-2 polypeptide or said variant is administered to said mammal in the form of a viral vector.

17. The method of claim 16, wherein said viral vector is an AAV vector.

18. The method of any one of claims 6-8, wherein said nucleic acid encoding said PC-2 polypeptide or said variant is operably linked to a promoter sequence.

19. The method of claim 18, wherein said promoter sequence is selected from the group consisting of a EFla promoter sequence, a CBh promoter sequence, a PKD1 promoter sequence, a PKD2 promoter sequence, a CMV promoter sequence, a RSV promoter sequence, an AQP2 promoter sequence, a Ggtl promoter sequence, and a Ksp-cadherin promoter sequence.

20. The method of claim 11, wherein said nucleic acid encoding said PC-1 polypeptide or said variant and said nucleic acid encoding said PC-2 polypeptide or said variant are administered to said mammal in the form of a viral vector.

21. The method of claim 20, wherein said viral vector is a HDAd vector.

22. The method of any one of claims 20-21, wherein said nucleic acid encoding said PC-1 polypeptide or said variant is operably linked to a first promoter sequence, and wherein said nucleic acid encoding said PC-2 polypeptide or said variant is operably linked to a second promoter sequence.

23. The method of claim 22, wherein said first promoter sequence and said second promoter sequence are each independently selected from the group consisting of a EFla promoter sequence, a CBh promoter sequence, a PKD1 promoter sequence, a PKD2 promoter sequence, a CMV promoter sequence, a RSV promoter sequence, an AQP2 promoter sequence, a Ggtl promoter sequence, and a Ksp-cadherin promoter sequence.

24. The method of any one of claims 1-23, wherein said method comprises identifying said mammal as being in need of a treatment for said PKD.

25. The method of any one of claims 1-24, wherein said mammal is a human.

26. The method of any one of claims 1-25, wherein PKD is an autosomal dominant PKD
(ADPKD).

27. The method of any one of claims 1-26, wherein said method further comprises, prior to said administering said nucleic acid, administering a lipopolysaccharides (LPS) to said mammal.

28. The method of claim 27, wherein said LPS is administered to said mammal at least 18 hours prior to said administering said nucleic acid.

29. The method of any one of claims 27-28, wherein administering said LPS
is effective to deliver large nucleic acid to said kidney cells in said mammal.

30. A method for treating a mammal having a PKD, wherein said method comprises administering to said mammal:
(a) nucleic acid encoding a fusion polypeptide including a deactivated Cas (dCas) polypeptide and a transcriptional activator polypeptide;
(b) nucleic acid encoding a helper activator polypeptide; and (c) nucleic acid encoding a nucleic acid molecule including (i) a nucleic acid sequence that is complementary to a target sequence within a PKD1 gene, and (ii) a nucleic acid sequence that can bind said helper activator polypeptide.

31. The method of claim 30, wherein said dCas polypeptide is selected from the group consisting of a deactivated Cas9 (dCas9) polypeptide, and a deactivated Cas phi (dCas(D) polypeptide.

32. The method of claim 30, wherein said transcriptional activator polypeptide is a VP64 polypeptide.

33. The method of any one of claims 30-32, wherein said fusion polypeptide is a dCas9-VP64 fusion polypeptide.

34. The method of any one of claims 30-33, wherein said helper activator polypeptide is selected from the group consisting of a MS2 polypeptide, a p65 polypeptide, a polypeptide, and a VP64 polypeptide.

35. The method of claim 34, wherein said helper activator polypeptide comprises a MS2 polypeptide, a p65 polypeptide, and a HSF1 polypeptide.

36. The method of any one of claims 30-35, wherein said nucleic acid (a), said nucleic acid (b), and said nucleic acid (c) are administered to said mammal in the form of a viral vector.

37. The method of claim 36, wherein said viral vector is selected from the group consisting of a HDAd, a lentiviral vector, and an AAV vector.

38. The method of any one of claims 30-35, wherein said nucleic acid (a) is administered to said mammal in the form of a first viral vector, and wherein said nucleic acid (b) and said nucleic acid (c) are administered to said mammal in the form of a second viral vector.

39. The method of claim 38, wherein said first viral vector is an AAV
vector.

40. The method of claim 38, wherein said second viral vector is an AAV
vector.

41. The method of any one of claims 30-40, wherein said nucleic acid (a) is operably linked to a first promoter sequence, said nucleic acid (b) is operably linked to a second promoter sequence, and said nucleic acid (c) is operably linked to a third promoter sequence.

42. The method of claim 41, wherein said first promoter sequence, said second promoter sequence, and said third promoter sequence are each independently selected from the group consisting of a EFla promoter sequence, a CBh promoter sequence, a CMV
promoter sequence, a RSV promoter sequence, a U6 promoter sequence, an AQP2 promoter sequence, a Ggtl promoter sequence, and a Ksp-cadherin promoter sequence.

43. The method of any one of claims 30-42, wherein said method comprises identifying said mammal as being in need of a treatment for said PKD.

44. The method of any one of claims 30-43, wherein said mammal is a human.

45. The method of any one of claims 30-44, wherein PKD is an ADPKD.

46. The method of any one of claims 30-35, wherein said method further comprises, prior to said administering said nucleic acid, administering a lipopolysaccharides (LPS) to said mammal.

47. The method of claim 46, wherein said LPS is administered to said mammal at least 18 hours prior to said administering said nucleic acid.

48. The method of any one of claims 46-47, wherein administering said LPS
is effective to deliver large nucleic acid to said kidney cells in said mammal.

49. A method for delivering nucleic acid to a cell within a mammal, wherein said method comprises:
(a) administering a proteinuria-inducing agent to said mammal; and (b) administering said nucleic acid to said mammal.

50. The method of claim 49, wherein said mammal is a human.

51. The method of any one of claims 49-50, wherein said proteinuria-inducing agent is selected from the group consisting of LPS, puromycin, adriamycin, protamine sulfate, cationic albumin, and polycations.

52. The method of any one of claims 49-51, wherein said nucleic acid is from about 0.15 kb to about 36 kb in size.

53. The method of any one of claims 49-51, wherein said nucleic acid has a mass of from about 10 kilodaltons (kDa) to about 50 kDa.

54. The method of any one of claims 49-51, wherein said nucleic acid has a diameter of from about 10 nm to about 26 nm.

55. The method of any one of clams 49-54, wherein said method comprises administering from about 7 milligrams per kilogram body weight (mg/kg) to about 9 mg/kg of said proteinuria-inducing agent to said mammal.

56. The method of any one of claims 49-55, wherein said cell is selected from the group consisting of a kidney cell, a spleen cell, a lungs cell, and a brain cell.

57. The method of any one of claims 49-56, wherein said proteinuria-inducing agent is administered to said mammal at least 18 hours prior to said administering said nucleic acid.

58. The method of any one of claims 49-57, wherein said administering said proteinuria-inducing agent comprises intravenous injection.

59. The method of any one of claims 49-57, wherein said administering said nucleic acid comprises intravenous injection.

60. The method of any one of claims 49-57, wherein said administering said proteinuria-inducing agent comprises intravenous injection, and wherein said administering said nucleic acid comprises intravenous injection.